tRNA Class Informative Features are Grounded in Physics, Highly Conserved, and Predictive of Functional Divergences of Human and Trypanosome aaRSs
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About This Presentation
Presented on June 15, 2022 by David H. Ardell at the tRNA 2022 meeting at The Ohio State University. Overview of Ardell laboratory research from Uppsala University and University of California, Merced.
Slide Content
tRNA Class Informative Features are Grounded in Physics,
Highly Conserved, and Predictive of Functional Divergences of
Human and Trypanosome aaRSs
David H. Ardell
Department of Molecular and Cellular Biology
University of California, Merced
tRNA 2022
The Ohio State Univ.
June 15, 2022
(Goodsell et al., 2015)
Highly Conserved, and Predictive of Functional Divergences of
Human and Trypanosome aaRSs
David H. Ardell
Department of Molecular and Cellular Biology
University of California, Merced
tRNA 2022
The Ohio State Univ.
June 15, 2022
(Goodsell et al., 2015)
Outline
tRNA CIFs Predict Functional
Divergences Between Human and
Trypanosome aaRSs
Rare tRNA CIFs are More
Functionally Informative
A B
A
B1
B2+3
C1
E
C3
F
G
scores Cyano-MLP
13
0.0
1.0
classification
probabilities0
1
2
3
4
bits
1
AA
SSMM
XX
JJ
FFNN
QQ
2
GG
JJ
CC
WW
YY
HH
3
GG
NN
VV
LL
CC
TT
EE
4
TT
JJ
EE
VV
AA
CC
RR
WW
HH
NN
MM
KK
YY
5
LL
GGKK
QQ
TT
JJ
FF
NN
AA
RR
YY
WW
II
6
QQ
VV
EE
MM
YY
JJ
KK
NN
HH
LL
RR
GG
AA
PP
DD
TT
7
SS
RR
GGNN
YY
KK
AA
VV
WW
JJ
II
89
GG
JJ
FF
10
SS
RRYY
11
RR
AA
NN
LL
TT
PP
QQ
YY
HHDD
EE
GG
WW
12
PP
JJSSWW
KK
VV
GG
MM
TT
NN
RR
AA
II
FF
DD
13
KK
YY
JJ
LL
CC
TT
AA
RR
QQ
MM
WW
GG
EE
14
SS
AA
YY
15
KK
QQJJ
16
PP
II
EE
KK
XX
HH
QQ
RR
SSNN
JJ
DD
WW
YY
VV
AA
TT
GG
FF
CC
MM
LL
17
SS
YY
HH
EE
QQ
NN
KK
TT
VV
RR
DD
FF
LL
WW
PP
AA
II
18
SS
TT
EEXX
PP
19 20 21
EE
JJ
YYLLDD
WW
KK
AA
TT
SS
MM
QQ
HH
XX
NN
FF
GG
PP
II
CC
VV
22
GG
PP
FFWW
NN
XX
CC
KK
EE
JJ
VV
SS
MM
HH
YY
DD
RR
II
23
PP
DD
NN
SS
RREE
24
YY
MM
JJ
LL
KK
25
KK
NN
FF
SS
QQYY
26
TTYY
KK
SS
27
MM
QQ
YYXX
28
SS
QQ
JJ
FF
EE
MM
VV
LL
AA
YY
TT
WW
KK
GG
RR
29
RR
LL
MM
NN
GG
WW
30
PP
VV
SS
LL
QQ
YY
JJ
FF
EE
WW
AA
MM
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TT
RR
CC
GG
XX
31
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WW
QQ
JJ
CC
EE
PP
TT
LL
DD
SS
GG
VV
FF
KK
NN
AA
HH
32
GGJJ
YY
DD
LL
MM
KK
SS
FF
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RR
AA
QQ
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PP
EE
33
AASS
34
VV
SS
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NN
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LL
GG
YY
XX
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QQ
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CC
WW
35
KK
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RR
AA
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LL
QQ
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36 37
SS
LL
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AA
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38
NN
QQ
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HH
DD
YY
KK
39
SS
RR
KK
TT
XX
MM
II
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NN
40AA
41
SSGG
RR
AA
PP
LL
VV
QQ
HH
42
NN
GG
QQ
EE
RR
AA
KK
HH
TT
YY
FF
LL
SS
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43
TT
GG
LL
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NN
AA
QQ
SS
MM
RR
YY
CC
JJ
44
FF
AA
NN
HH
JJ
KK
XX
MM
YY
RR
PP
VV
SS
LL
GG
45
KK
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PP
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HH
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YY
TT
FF
CC
WW
II
46
QQ
JJ
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MM
GG
YY
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WW
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LL
SS
KK
NN
47
AAQQ
FF
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WW
RR
JJ
EE
GG
MM
HH
CC
LL
YY
KK
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SS
48
TT
KK
HH
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NN
AA
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CC
MM
PP
VV
GG
RR
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WW
49
RR
KK
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50
YY
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51
SS
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YY
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AA
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WW
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QQ
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LL
52
GG
II
KK
53
VV
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AA
CC
MM
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GG
QQ
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WW
YY
TT
SS
FF
PP
HH
NN
54
QQ
TT
MM
LL
PP
NN
JJ
VV
XX
YY
II
55
GG
NN
YY
56 57 58 59YY
60 61 62
AA
RRDD
EE
CC
XX
KK
LL
QQ
MM
YY
FF
NN
TT
GG
HH
JJ
63
DD
QQ
FF
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YY
AA
HH
NN
RR
MM
EE
JJ
LL
KK
VV
GG
II
XX
SS
CC
PP
WW
64
VVYYEE
65
RR
VV
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NN
AA
GG
YY
XX
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WW
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CC
66
AA
SS
YY
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VV
RR
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QQ
67
GG
MM
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68
SS
NNMM
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LL
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69
RRSS
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AA
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70
KK
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71
YY
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SS
72
SS
YY
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GG
AA
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JJ
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MM
HH
FF
DD
73
RRTT
NN
JJ
WW
YY
VV
AA
SS
74
YY
DD
NN
XX
WW
RR
75
TT
GGWW
NN
VV
RR
MM
LL
YY
XX
EE
76
SS
KK
QQ
JJ
MM
EE
YYGG
TT
CC 0
1
2
3
4
bits
1
RR
PP
SS
LL
KK
EE
FF
MM
JJ
XXNN
QQ
2
SS
PPJJ
II
MM
WW
CC
HH
3
SS
VV
EE
RR
FF
JJ
CC
II
MM
LL
TT
4
LL
SS
FF
WW
AA
II
QQ
VV
CC
RR
HH
JJNN
MM
KK
YY
5
PP
SS
QQ
DD
HH
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NN
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AA
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II
6
SS
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HH
QQ
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JJ
VV
LL
RR
GG
PP
TT
AA
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7
LL
RR
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QQ
YY
SS
NN
MM
KK
VV
AA
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II
89
10
WWSSYY
11
VV
SS
LL
HH
PP
RR
QQEE
GG
DD
WW
12
EE
CC
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SS
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AA
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13
PP
AA
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14 15
AA
SS
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16
II
PP
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17
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II
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AA
18
TT
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XX
19 20 21
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QQ
NN
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28
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29
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34
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35
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36 37
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43
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45
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46
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47
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48
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XX
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49
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56
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71
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72
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74
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75
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76
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CC 0
1
2
3
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bits
1
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23
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4
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36 37
FF
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38
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39
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40
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41
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42
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55
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56 57 58 59 60 61 62
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63
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64
PPAAEE
65
PP
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66
SSLL
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67
AA
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68
KKSS
NN
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MM
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HH
69
KK
DD
II
RR
WW
TT
NN
MM
LL
SS
JJ
GG
VV
AA
YY
CC
FF
XX
EE
70
SS
EE
DDWW
CCTT
GG
XX
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QQ
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MM
AA
VV
NN
II
71
EE
NN
II
VV
KK
QQ
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AA
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HH
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72
AA
GGSS
EE
NN
CC
XX
II
KK
MM
JJ
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TT
PP
FF
HH
DD
73
LL
PP
GGKK
EE
NN
WW
CC
TT
II
RR
MM
VV
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SS
AA
74
SS
TT
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II
EE
NN
DD
RR
75
PP
VV
NN
LL
MM
JJ
RR
WW
XXEE
76
RRKKGG
TT
CC 0
1
2
3
4
bits
1
AAMMNN
QQ
2
EEMM
3
WWRR
4
VV
EE
RRWWMM
KK
YY
NN
5
AAII
LL
RR
TT
6
HH
MM
DD
PP
LL
AA
GG
TT
RR
JJ
7
GG
SSKKRR
TT
AA
WW
II
VV
89
VV
HH
GG
10 11
CCSS
LL
GG
EE
WW
DD
12
PPSS
VV
GG
WW
II
MM
RR
NN
KK
TT
DD
AA
FF
13
VVGG
EE
14
WWJJ
15 16
MM
NN
KK
TT
VV
PP
DD
RR
AA
QQ
XX
WW
CC
SS
HH
LL
GG
JJ
YY
FF
17
EETTDD
RR
LL
MM
WW
II
FF
PP
AA
18XX
PP
19 20 21
CC
WW
TT
VV
GG
QQ
JJ
NN
DD
KK
XX
PP
MM
YY
FF
II
SS
LL
AA
HH
22
CCSS
HH
II
YY
MM
RR
DD
23
SS
RREE
24
LL
25
VVSSYY
26
CC
HH
SSJJ
27
WWXX
28
LLGG
29SS
30
VVMM
QQ
RR
KK
XX
GG
31
HH
YY
AA
SS
PP
LL
TT
KK
VV
NN
32
LLTT
SS
GG
RR
PP
VV
AA
EE
33
PP
AA
HH
34
VVRR
SS
PP
TT
LL
WW
35
SS
EE
WW
FFAA
RR
LL
VV
GG
CC
TT
HH
QQ
MM
PP
36 37
RR
SS
TT
GG
PP
VV
LL
AA
KK
QQ
EE
38
KK
QQ
YY
DD
NN
HH
EE
39
SS
RR
II
KK
TT
XX
MM
JJ
NN
40 41
MM
VVRR
HH
AA
LL
PP
QQ
42
WW
HHVV
TTLL
SS
RR
NN
CC
JJ
YY
FF
43
LLQQ
JJ
44
MMSS
LL
JJ
RR
PP
VV
GG
45
LL
MM
SS
WW
II
CC
46
LL
PP
KK
TT
SS
RR
CC
NN
47
TTVV
GG
HH
LL
SS
KK
YY
NN
CC
48
RR
PP
GG
VV
AA
JJ
WW
FF
49
SS
LL
JJHH
YY
QQ
50
EE
LLSS
GG
PP
HH
TT
CC
VV
RR
AA
QQ
DD
NN
KK
XX
MM
FF
II
WW
51
EEGGDD
LL
52
RRTT
53
VVLL
RR
PP
TT
SS
KK
QQ
YY
54
XX
TT
RRVV
II
YY
55
RR
CC
WWTT
MM
56 57 58 59 60 61 62
RREETT
VV
GG
JJ
63
VV
DD
EE
HH
GG
QQ
RR
NN
KK
FF
MM
YY
II
CC
TT
SS
WW
XX
LL
AA
PP
64 65
RR
TT
AA
GG
LL
MM
KK
JJ
XX
CC
FF
66
NN
RR
XX
VVPP
WW
QQ
67
LL
PP
EE
CCMM
TT
GG
SS
RR
68
RRLLXXPP
VV
GG
TT
HH
EE
AA
DD
69
PPTT
SS
LL
MM
AA
EE
CC
FF
70
TTWW
PPAA
MMVV
II
71
MM
AA
HH
TT
RR
PP
GG
LL
SS
72
RRLL
PP
MM
HH
FF
DD
73
LLAA
SS
74
MMRR
75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
HHQQ
NN
23
LL
EE
TT
4
RR
JJ
TTNN
WW
KK
MM
YY
5
LL
TT
FF
KK
II
6
NN
VV
MM
LL
PP
TT
RR
QQ
AA
DD
GG
7
RR
KK
AA
JJ
YY
VV
WW
II
89
10 11
QQ
NN
LL
GG
DD
EE
WW
12
JJ
SSVV
AA
RR
TT
FF
GG
II
DD
KK
WW
MM
NN
13
RR
QQ
GG
EE
14 15 16 17
HH
EE
NN
TT
QQ
RR
DD
FF
LL
VV
PP
II
WW
AA
18
JJXX
PP
19 20 21
E
J
Q
H
N
LL
T
F
II
C
W
A
G
K
D
Y
S
P
M
V
X
22
VV
MM
JJ
RR
SS
HH
DD
YY
II
23
RR
DDEE
24
RR
LLJJ
25
SSYY
26SS
27
QQ
JJXX
28
LL
QQ
DD
29 30
SS
QQ
RR
KK
JJ
WW
GG
XX
31
AA
VV
SS
YY
TT
GG
PP
LL
FF
KK
NN
HH
32
LLGG
FF
QQ
DD
RR
NN
SS
TT
PP
EE
VV
AA
33 34
R
S
T
F
H
DD
KK
CC
PP
WW
35
NN
RR
VV
QQ
AA
GG
LL
CC
HH
TT
PP
36 37
SSPP
GG
LL
RR
TT
VV
AA
KK
QQ
EE
38
KK
HH
DD
EE
YY
NN
QQ
39
SS
RR
TT
XX
MM
KK
NN
JJ
II
40 41
RR
AA
PP
LL
VV
QQ
HH
42
AA
EE
RR
QQ
NN
TT
JJ
LL
SS
CC
HH
FF
YY
43
RR
QQ
JJ
44
VV
RR
SS
PP
LL
GG
45
GG
LL
TT
VV
EE
SS
WW
FF
II
CC
46
LL
JJ
QQ
RR
SS
MM
KK
CC
NN
47
JJ
HH
EE
GG
KK
MM
CC
NN
LL
YY
SS
48
JJ
LL
PP
RR
VV
AA
GG
FF
WW
49
JJ
SS
EE
LL
CC
QQ
HH
YY
50 51
RR
TT
EE
NN
GG
QQ
DD
LL
52 53
RR
JJ
FF
GG
LL
HH
TT
PP
YY
NN
KK
QQ
54
PP
RR
JJ
VV
XX
II
YY
55 56 57 58 59 60 61 62
LLTT
JJ
GG
63 64EE
65
RR
AA
GG
DD
LL
TT
CC
66
RR
TT
LL
KK
PP
JJ
WW
QQ
NN
67
GG
JJ
LL
WW
RR
SS
XX
68
FF
RR
EE
NN
LL
MM
PP
GG
DD
VV
TT
HH
AA
69
LL
TT
SS
GG
VV
AA
EE
FF
CC
70
RR
SS
AA
JJ
FF
QQ
LL
VV
YY
MM
NN
II
71
AA
GG
RR
TT
LL
PP
HH
SS
72
SS
TT
RR
PP
FF
HH
73
RR
JJ
VV
SS
AA
74RR
75
RR
LL
WWEE
76
QQTT
CC
GG 0
1
2
3
4
bits
1
GG
TT
FF
JJ
MM
HH
QQ
NN
2
RRFF
HH
CC
3
GG
VV
QQ
LL
TT
EE
4
JJ
WW
NN
MM
KK
YY
5
VV
TT
EE
KK
WW
RRII
6
FF
VV
MM
WW
EE
NN
LL
QQ
GG
RR
AA
PP
TT
DD
7
GG
AA
KK
VV
WW
JJ
II
89
10
HH
11
RR
LL
QQ
GG
EE
DD
WW
12
SS
JJ
WW
VV
RR
KK
AA
GG
TT
II
MM
FF
DD
NN
13
QQ
RR
WW
YY
GG
EE
14 15 16
PPKK
JJEE
QQ
VV
RR
DD
HH
CC
FF
XX
GG
NN
AA
TT
LL
WW
MM
YY
SS
17
SS
JJ
RR
KK
QQ
TT
FF
MM
VV
DD
LL
WW
II
PP
AA
18
VVPP
XX
19 20 21 22
TT
CC
EE
VV
SSDD
HH
YY
RR
MM
II
JJ
23
DD
RR
EE
24
RR
VV
LL
KK
YY
25
SS
FF
NN
YY
26
KKSS
27
VV
EE
WW
QQXX
28
RR
GG
VV
KK
QQ
AA
29
VV
LL
QQ
30
LL
TT
SS
PP
VV
AA
JJ
MM
QQ
CC
RR
WW
GG
XX
31
WW
GG
SS
RR
DD
AA
PP
EE
VV
TT
FF
LL
KK
HH
NN
32
LL
WW
SS
YY
FF
RR
TT
QQ
VV
AA
PP
EE
33
VV
FF
34
RR
GG
VV
TT
QQ
FF
PP
LL
CC
WW
35
FF
RR
SS
EE
AA
QQ
GG
LL
VV
TT
PP
HH
36 37
SS
GG
RR
PP
VV
LL
AA
TT
KK
QQ
EE
38
DD
EE
HH
KK
YY
NN
QQ
39
SS
RR
II
KK
JJ
TT
XX
NN
MM
40 41
GG
RR
PP
VV
AA
LL
QQ
HH
42
VV
KK
EE
AA
MM
QQ
NN
RR
LL
TT
FF
HH
YY
JJ
SS
43
GG
RR
CC
JJ
44
TT
KK
JJ
MM
RR
WW
PP
VV
HH
SS
LL
GG
45
LL
SS
GG
TT
WW
FF
CC
II
46
GG
WW
LL
TT
YY
RR
SS
KK
CC
NN
47
TT
RR
GG
VV
WW
EE
HH
LL
YY
NN
KK
SS
CC
48
SS
LL
RR
PP
VV
AA
GG
WW
FF
JJ
49
SS
RR
KK
LL
CC
JJ
QQ
YY
HH
50 51
SS
VV
RR
TT
QQ
WW
EE
GG
YY
KK
DD
LL
52
VVKK
53
SS
VV
RR
MM
WW
CC
NN
TT
PP
LL
QQ
GG
HH
KK
54
RR
NN
VV
XX
YY
II
55 56 57 58 59 60 61 62
LL
KK
RR
TT
HH
YY
NN
GG
FF
63 64
AAEE
65
SS
GG
NN
YY
LL
MM
TT
CC
FF
66
LLGG
FF
SS
VV
RR
MM
YY
TT
KK
PP
NN
JJ
WW
QQ
67
GG
VV
LL
QQ
SS
RR
XX
68
RR
LL
SS
JJ
MM
FF
GG
PP
VV
EE
TT
DD
HH
AA
69
SS
WW
VV
GG
EE
AA
CC
FF
70
TT
KK
GG
JJ
AA
QQ
RR
LL
DD
VV
MM
NN
II
71
FF
JJ
GG
MM
RR
WW
CC
AA
VV
TT
LL
HH
PP
SS
72
GG
TTJJ
MM
RR
PP
HH
FF
DD
73
RR
MM
VV
JJ
SS
AA
74
WW
RR
75
RR
WW
LL
YYEE
76
VV
EE
WW
QQGG
TT
CC 0
1
2
3
4
bits
1
JJ
DDQQ
NN
2HH
3
GG
VV
EE
LL
MM
DD
JJ
TT
4
AA
FF
RR
DD
CC
HH
WW
NN
KK
YY
5
AA
KK
LL
FF
HH
EE
TT
NN
WW
RR
DD
II
6
VV
KK
MM
NN
LL
QQ
EE
HH
CC
DD
TT
AA
RR
GG
PP
7
SS
KK
EE
AA
MM
JJ
VV
WW
II
89
10 11
RR
PP
LL
HH
GG
DD
EE
WW
12
PP
SS
KK
JJ
TT
AA
GG
WW
VV
RR
II
MM
NN
FF
DD
13
LL
SS
AAWW
DD
EE
GG
14 15 16
PP
II
EE
MM
KK
TT
XX
NN
QQ
DDHH
YY
CC
VV
AA
FF
JJ
LL
GG
RR
SS
WW
17
SS
KK
EE
TT
QQMM
RR
LL
DD
VV
PP
FF
WW
AA
II
18
RRJJ
PP
XX
19 20
DD
RR
21 22
GG
KK
EE
CC
VV
SS
MM
RR
HH
DD
II
YY
23
SSRR
DD
CCEE
24
SS
LL
25
SS
FF
NNYY
26
KKTT
SS
27
QQ
EE
JJ
XX
28
LLGG
FF
AA
VV
KK
29
LL
GG
30
LL
EE
PP
QQ
WW
SS
RR
AA
TT
JJ
VV
KK
GG
XX
31
PP
RR
EE
TT
JJ
FF
YY
SS
AA
LL
VV
HH
KK
NN
32
SS
YY
CC
LL
RR
FF
QQ
VV
AA
TT
PP
GG
EE
33
SS
LL
MM
34
GG
RR
QQ
YY
LL
SS
TT
EE
PP
FF
HH
CC
MM
WW
35
SS
RR
EE
AA
LL
GG
QQ
HH
VV
TT
PP
36 37
SS
RR
TT
GG
LL
VV
AA
KK
PP
QQ
EE
38
KK
YY
NN
EE
QQ
HH
DD
39
SS
RR
XX
MM
KK
NN
JJ
TT
II
40 41
RR
AA
PP
VV
LL
QQ
HH
42
GG
RR
EE
HH
AA
TT
KK
NN
JJ
LL
FF
SS
YY
MM
43
AA
LL
HH
44
QQ
MM
JJ
AA
WW
RR
PP
SS
GG
CC
VV
LL
45
PP
EE
GG
LL
MM
YY
SS
JJ
FF
HH
WW
CC
II
46
EE
GG
VV
LL
TT
SS
RR
NN
JJ
KK
CC
MM
47
VV
MM
HH
WW
QQ
GG
NN
CC
LL
YY
SS
KK
48
AA
LL
KK
HH
PP
VV
GG
RR
FF
WW
49
KK
MM
LL
CC
JJ
YY
HH
QQ
50
S
LL
CC
EE
JJ
AA
GG
QQ
MM
NN
KK
PP
TT
RR
FF
WW
HH
II
VV
XX
DD
51
SS
RR
TT
VV
AA
NN
HH
WW
EE
QQ
MM
GG
JJ
KK
DD
LL
52
EE
PP
53
LLAA
EE
GG
QQ
RR
YY
CC
TT
II
FF
JJ
NN
KK
MM
HH
PP
54
LL
PP
KK
RR
CC
NN
VV
JJ
XX
YY
II
55 56 57 58 59 60 61 62
SS
TT
LL
KK
RR
PP
NN
QQ
FF
MM
HH
GG
JJ
63 64
AAEE
65
SS
RR
KK
EE
NN
DD
JJ
AA
MM
TT
GG
LL
FF
CC
66
SS
VV
TT
JJ
RR
LL
NN
PP
WW
QQ
67
PP
WW
JJ
RR
VV
GG
LL
SS
QQ
68
SS
KK
RR
II
CC
LL
JJ
EE
FF
PP
VV
GG
AA
TT
HH
DD
XX
69
GGKK
RR
DD
WW
CC
TT
SS
LL
VV
MM
EE
FF
AA
YY
70
WW
CC
AA
LL
RR
QQ
VV
FF
KK
NN
II
71
KK
QQ
AA
CC
VV
TT
RR
GG
LL
HH
PP
SS
72
EE
GG
RR
JJ
PP
DD
HH
FF
MM
73
PP
JJ
WW
VV
AA
SS
74
VVRR
75
RR
VV
LLMM
EE
76
SS
KKTT
GG
CC 0
1
2
3
4
bits
1NN
QQ
23
NNTT
CC
4
RRMM
NN
WW
KK
YY
5
KK
VV
RR
LL
EEII
6
VV
LLRR
DD
GG
PP
AA
TT
7
KK
AA
VV
JJ
II
WW
89
10
SS
YY
11
SS
LL
QQDD
GG
WW
EE
12
JJ
SSVV
TT
RR
DD
GG
FF
AA
WW
II
MM
KK
NN
13
RR
QQ
GG
EE
14 15 16
PPKK
NN
MM
HH
QQ
AA
VV
RR
JJ
LL
SS
CC
YY
DD
TT
GG
FF
WW
17
RR
QQ
NN
VV
LL
TT
WW
DD
AA
FF
II
PP
18
RRJJPP
XX
19 20 21
DD
QQ
NN
LL
AA
VV
TT
FF
GG
XX
CC
WW
HH
KK
MM
YY
PP
II
SS
JJ
22
AA
VV
SS
JJ
RR
DD
II
HH
YY
23
SS
RR
EE
24
GG
LLJJ
25
RRSS
26
RR
GGSS
27
JJXX
28
GG
SS
DD
VV
29
RRLL
MM
GG
30
SS
RR
TT
QQ
JJ
HH
PP
KK
GG
WW
XX
31
DD
RR
TT
VV
GG
PP
LL
AA
KK
NN
32
LLKK
QQ
SS
RR
TT
GG
AA
PP
VV
EE
33
VV
SS
34
TT
LL
GG
PP
SS
CC
WW
35
SSRR
MM
LL
AA
TT
GG
VV
HH
QQ
PP
36 37
SS
RR
GG
TT
PP
VV
LL
AA
EE
QQ
KK
38
HH
YY
QQ
DD
KK
NN
EE
39
SS
RR
II
XX
MM
NN
TT
KK
JJ
40 41
VV
RR
LL
AA
PP
HH
QQ
42
RR
KK
SS
AA
NN
TT
LL
JJ
FF
HH
YY
43
PP
RRJJ
44
RR
TT
SS
QQ
PP
LL
VV
JJ
GG
45
AA
GG
PP
SS
WW
LL
EE
CC
HH
FF
II
46
TT
GG
JJ
CC
YY
LL
RR
SS
NN
KK
MM
47
RR
GG
HH
CC
MM
SS
LL
NN
KK
YY
48
KK
GG
VV
RR
JJ
FF
WW
49
RR
LL
KKJJ
QQ
HH
CC
YY
50
LL
KK
JJ
CCVV
RR
QQ
PP
GG
FF
DD
AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
QQ
JJ
GG
DD
LL
52 53
RR
KK
LL
QQ
NN
PP
TT
GG
SS
HH
YY
54
RR
NN
JJ
VV
QQ
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
RRTT
KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
QQ
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
QQ
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
QQ
FF
EE
70
RR
VV
QQ
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
genera
Oscillatoria
Arthrospira
Anabaena
Nostoc
Cyanothece
Spirulina
clades
Pleurocapsa
Prochlorococcus
Gloeobacter
Pseudanabaena
Acaryochloris
Nodosilinea
function logos
Genome
g
tRNA
genes
T
g
C
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g
Nonetheless, tRNA CIFs are Highly
Evolutionarily Conserved
tRNA Class Informative Features
(CIFs) are Grounded in Physics,
not Evolutionary Conservation
tRNA CIFs Predict Functional
Divergences Between Human and
Trypanosome aaRSs
Rare tRNA CIFs are More
Functionally Informative
A B
A
B1
B2+3
C1
E
C3
F
G
scores Cyano-MLP
13
0.0
1.0
classification
probabilities0
1
2
3
4
bits
1
AA
SSMM
XX
JJ
FFNN
2
GG
JJ
CC
WW
YY
HH
3
GG
NN
VV
LL
CC
TT
EE
4
TT
JJ
EE
VV
AA
CC
RR
WW
HH
NN
MM
KK
YY
5
LL
GGKK
TT
JJ
FF
NN
AA
RR
YY
WW
II
6
VV
EE
MM
YY
JJ
KK
NN
HH
LL
RR
GG
AA
PP
DD
TT
7
SS
RR
GGNN
YY
KK
AA
VV
WW
JJ
II
89
GG
JJ
FF
10
SS
RRYY
11
RR
AA
NN
LL
TT
PP
YY
HHDD
EE
GG
WW
12
PP
JJSSWW
KK
VV
GG
MM
TT
NN
RR
AA
II
FF
DD
13
KK
YY
JJ
LL
CC
TT
AA
RR
MM
WW
GG
EE
14
SS
AA
YY
15
KK
QQJJ
16
PP
II
EE
KK
XX
HH
RR
SSNN
JJ
DD
WW
YY
VV
AA
TT
GG
FF
CC
MM
LL
17
SS
YY
HH
EE
NN
KK
TT
VV
RR
DD
FF
LL
WW
PP
AA
II
18
SS
TT
EEXX
PP
19 20 21
EE
JJ
YYLLDD
WW
KK
AA
TT
SS
MM
HH
XX
NN
FF
GG
PP
II
CC
VV
22
GG
PP
FFWW
NN
XX
CC
KK
EE
JJ
VV
SS
MM
HH
YY
DD
RR
II
23
PP
DD
NN
SS
RREE
24
YY
MM
JJ
LL
KK
25
KK
NN
FF
SS
QQYY
26
TTYY
KK
SS
27
MM
YYXX
28
SS
JJ
FF
EE
MM
VV
LL
AA
YY
TT
WW
KK
GG
RR
29
RR
LL
MM
NN
GG
WW
30
PP
VV
SS
LL
YY
JJ
FF
EE
WW
AA
MM
NNKK
TT
RR
CC
GG
XX
31
RRYY
WW
JJ
CC
EE
PP
TT
LL
DD
SS
GG
VV
FF
KK
NN
AA
HH
32
GGJJ
YY
DD
LL
MM
KK
SS
FF
TT
RR
AA
VV
PP
EE
33
AASS
34
VV
SS
JJ
EE
AA
MM
NN
TT
RR
DD
LL
GG
YY
XX
PP
FF
HH
CC
WW
35
KK
YYNN
FF
EE
MM
SS
RR
AA
GG
LL
VV
TT
HH
PP
36 37
SS
LL
TT
GG
PP
VV
RR
AA
KK
EE
38
NN
EE
HH
DD
YY
KK
39
SS
RR
KK
TT
XX
MM
II
JJ
NN
40AA
41
SSGG
RR
AA
PP
LL
VV
HH
42
NN
GG
EE
RR
AA
KK
HH
TT
YY
FF
LL
SS
JJ
43
TT
GG
LL
EE
DD
NN
AA
SS
MM
RR
YY
CC
JJ
44
FF
AA
NN
HH
JJ
KK
XX
MM
YY
RR
PP
VV
SS
LL
GG
45
KK
GG
PP
DD
MM
HH
RR
SS
EE
LL
YY
TT
FF
CC
WW
II
46
JJ
FF
MM
GG
YY
RR
WW
TT
CC
LL
SS
KK
NN
47
AAQQ
FF
DDTT
WW
RR
JJ
EE
GG
MM
HH
CC
LL
YY
KK
NN
SS
48
TT
KK
HH
SS
NN
AA
JJ
LL
CC
MM
PP
VV
GG
RR
FF
WW
49
RR
KK
FF
EE
SS
JJ
CC
HH
LL
YY
50
YY
JJ
SS
LL
EE
CC
GGQQMM
WW
AA
KK
RR
FF
TT
NN
VV
PP
XX
II
DD
HH
51
SS
PP
FF
CC
HH
JJ
VV
YY
TT
AA
NN
RR
MM
WW
EE
GG
KK
DD
LL
52
GG
II
KK
53
VV
JJ
AA
CC
MM
LL
KK
GG
RR
WW
YY
TT
SS
FF
PP
HH
NN
54
TT
MM
LL
PP
NN
JJ
VV
XX
YY
II
55
GG
NN
YY
56 57 58 59YY
60 61 62
AA
RRDD
EE
CC
XX
KK
LL
MM
YY
FF
NN
TT
GG
HH
JJ
63
DD
FF
TT
YY
AA
HH
NN
RR
MM
EE
JJ
LL
KK
VV
GG
II
XX
SS
CC
PP
WW
64
VVYYEE
65
RR
VV
SS
NN
AA
GG
YY
XX
DD
WW
FF
TT
LL
CC
66
AA
SS
YY
FF
MM
XX
TT
LL
JJ
VV
RR
KK
PP
WW
NN
67
GG
MM
KK
YY
WW
LL
SS
RR
68
SS
NNMM
FF
KK
LL
JJ
GG
PP
TT
EE
XX
VV
AA
HH
DD
69
RRSS
JJ
TT
MMLL
KK
DD
NN
WW
YY
VV
GG
XX
AA
EE
FF
CC
70
KK
SS
GG
WW
NN
LL
RR
JJ
DD
AA
MM
FF
VV
II
71
YY
FF
NN
KK
JJ
WW
AA
CC
TT
GG
RR
LL
VV
HH
PP
SS
72
SS
YY
TT
GG
AA
NN
XXKK
JJ
RR
PP
MM
HH
FF
DD
73
RRTT
NN
JJ
WW
YY
VV
AA
SS
74
YY
DD
NN
XX
WW
RR
75
TT
GGWW
NN
VV
RR
MM
LL
YY
XX
EE
76
SS
KK
JJ
MM
EE
YYGG
TT
CC 0
1
2
3
4
bits
1
RR
PP
SS
LL
KK
EE
FF
MM
JJ
XXNN
2
SS
PPJJ
II
MM
WW
CC
HH
3
SS
VV
EE
RR
FF
JJ
CC
II
MM
LL
TT
4
LL
SS
FF
WW
AA
II
VV
CC
RR
HH
JJNN
MM
KK
YY
5
PP
SS
DD
HH
GG
KK
RR
LL
NN
EE
AA
FFWW
II
6
SS
WW
CC
KK
NN
MM
HH
EE
JJ
VV
LL
RR
GG
PP
TT
AA
DD
7
LL
RR
TT
YY
SS
NN
MM
KK
VV
AA
WW
JJ
II
89
10
WWSSYY
11
VV
SS
LL
HH
PP
RR
QQEE
GG
DD
WW
12
EE
CC
PP
SS
JJ
WW
TT
MM
RR
KK
GG
VV
FF
AA
II
NN
DD
13
PP
AA
SSNN
DD
CC
KK
MM
JJ
YY
RR
WW
GG
EE
14 15
AA
SS
NN
YY
KK
MM
XX
JJ
16
II
PP
XX
EE
KK
JJ
WW
LL
MM
HH
RR
NN
YY
DD
VV
SS
TT
AA
GG
CC
FF
17
SS
TTNN
EE
KK
YY
HH
JJ
RR
MM
WW
VV
DD
FF
II
LL
PP
AA
18
TT
II
FF
JJ
YYMMPP
XX
19 20 21
EE
LL
JJ
NN
WW
MM
KK
AA
DD
SS
GG
TT
PP
CC
YY
VV
XX
FF
II
HH
22
LL
GG
KK
WW
AA
XX
CC
EEVV
SS
JJ
MM
RR
HH
YY
II
DD
23
LLPP
SS
DD
YY
RR
EE
24
SSRR
JJ
YY
KK
LL
25
QQFF
SSYY
26
VV
YYKK
TT
SS
27
JJMM
EEXX
28
PP
TTEE
YYCC
DD
AA
RR
LL
VV
SS
JJ
WW
MM
GG
KK
29
RR
SS
PP
VV
WW
KK
LL
GG
30
LL
NN
JJ
AA
FF
DD
MM
EE
KK
CC
SS
WW
RR
TT
GG
XX
31
MM
II
FFQQ
WW
RR
SS
TT
DD
CC
JJ
EE
PP
AA
GG
VV
LL
KK
HH
NN
32
WW
JJ
YY
HH
IIGG
DD
SS
LL
KK
TT
RR
FF
VV
PP
AA
EE
33
LL
GG
EE
SS
34
AA
MM
KK
SS
VVGG
XX
RR
DD
EE
LL
YY
FF
PP
HH
WW
CC
35
KK
II
NN
CC
MM
XX
WW
DD
EE
FF
SS
RR
VV
AA
GG
LL
TT
HH
PP
36 37
IISS
RR
LL
TT
PP
GG
VV
KK
AA
EE
38
YY
NN
DD
HH
KK
EE
39
SS
RR
MM
XX
TT
JJ
KK
NN
II
40DD
41
SS
EEGG
AA
RR
PP
VV
LL
HH
42
PP
XX
II
WW
EE
MM
AA
RR
HH
KK
NN
LL
TT
FF
JJ
YY
SS
43
II
KK
FF
CC
RR
HH
SS
MM
JJ
44
AA
CC
HH
WW
YY
MM
KK
JJ
XX
SS
PP
RR
VV
LL
GG
45
KK
NN
EE
RR
PP
JJ
VV
GG
LL
SS
HH
TT
MM
FF
WW
CC
II
46
PP
II
VV
EE
DDLL
TT
WW
GG
MM
CC
SS
JJ
RR
YY
KK
NN
47
TTMM
RR
HH
JJ
WW
GG
CC
EE
LL
NN
KK
YY
SS
48
IITT
YY
XX
DD
NN
CC
MM
LL
KK
RR
PP
JJ
SS
GG
VV
AA
WW
FF
49
PP
II
RR
KK
SS
MMJJ
LL
HH
CC
YY
50
SS
EE
YY
CC
LL
JJ
PP
GG
MM
KK
RR
WW
TT
HH
XX
DD
FF
NN
AA
II
VV
51
PP
CCHH
SS
VV
FF
AA
TT
MM
EE
YY
RR
JJ
NN
WW
GG
KK
DD
LL
52
LL
AASS
PP
53
EE
YY
AA
LL
XXCC
DD
SS
RR
JJ
WW
GG
VV
FF
KK
PP
NN
TT
MM
HH
54
SS
TT
FF
WW
LL
JJ
PP
NN
MM
VV
YY
XX
II
55
SSJJMM
56
LL
VV
WW
57 58 59
SS
RR
60 61 62
RR
DD
PP
SS
VV
AA
NN
YY
MM
EE
CC
KK
HH
LL
JJ
FF
GG
TT
63
DD
TT
RR
FF
KK
HH
AA
CC
YY
WW
SS
NN
PP
LL
GG
JJ
EE
VV
MM
XX
II
64
LL
YY
AAEE
65
PP
JJ
HHDD
XX
RR
GG
YY
NN
KK
EE
MM
SS
VV
AA
CC
LL
TT
FF
66
EE
FF
II
SS
XX
TT
MM
VV
YY
KK
LL
RR
PP
NN
JJ
WW
67
AA
PP
YY
MM
WW
XX
KK
LL
SS
RR
68
II
RR
SS
NN
MM
KK
JJ
EE
LL
TT
PP
GG
FF
XX
VV
AA
HH
DD
69
KK
QQDDJJHH
RR
LL
MM
SS
NN
TT
AA
WW
YY
VV
GG
EE
FF
CC
XX
70
PPHH
EEVV
CC
SS
JJ
AA
RR
LL
FF
KK
NN
MM
II
71
GG
EE
KK
NN
RR
WW
DD
JJ
AA
VV
TT
CC
PP
LL
HH
SS
72
LL
AA
SS
WW
GG
KK
NN
MM
JJ
TT
RR
PP
FF
DD
HH
73
TT
II
RR
KK
MM
NN
WW
VV
JJ
SS
AA
74
SS
PP
KK
VV
EE
WW
JJDD
XXRR
75
KK
SS
XX
NN
VV
WW
RR
JJ
MM
LLEE
76
RR
LLKK
FF
SS
XX
EETT
GG
CC 0
1
2
3
4
bits
1
AA
GG
VV
KKNN
23
LL
RR
VV
FF
EE
CC
TT
4
AA
GG
VV
LL
CC
DD
HH
TT
JJ
RR
WW
MM
KK
NN
YY
5
LLJJ
EE
NN
YY
KK
AA
TT
MM
DD
RR
WW
II
6
SSII
JJ
CC
MM
KK
EE
WW
HHLL
VV
RR
TT
PP
AA
GG
DD
7
SS
PP
LL
RR
NN
KK
QQMM
TT
AA
VV
JJ
WW
II
89
10
SSYY
11
SS
AA
PP
RR
HH
TTLLGG
DD
EE
WW
12
PPEEJJ
SSMM
VV
WW
TT
GG
NN
DD
KK
RR
AA
FF
II
13
SS
KK
JJ
DD
HH
TT
MM
WW
RR
GG
EE
14 15 16
PP
II
JJKK
EE
DD
VV
NN
RR
HH
SS
XX
MM
TT
CC
FF
GG
YY
WW
AA
LL
17
SSNN
CC
EE
YY
JJ
KK
RR
TT
VV
MM
LL
DD
FF
II
PP
WW
AA
18
FFJJXX
PP
19 20 21
R
EETT
KK
WW
LL
NN
VV
GG
PP
AA
II
SS
YY
JJ
DD
FF
XX
CC
HH
MM
22
TT
PP
GG
EE
CCVV
SS
JJ
MM
RR
HH
II
DD
YY
23
PP
DD
SSRREE
24
LL
MM
JJ
25
TT
PPHH
SSYY
26
PP
GG
TT
JJ
MMSS
27
AA
PP
KK
EE
JJXX
28
SS
LL
JJ
EE
NN
GG
PP
WW
RR
TT
KK
DD
AA
VV
29
RR
PP
EE
NN
GG
TT
VV
LL
30
PP
LL
II
EE
VV
FF
CC
SS
MMTT
DD
AA
JJ
KK
RR
WW
GG
XX
31
EE
XX
RR
JJ
IIYY
CC
FF
SS
VV
DD
TT
GG
AA
PP
LL
HH
KK
NN
32
LLGG
II
KK
NN
CC
YY
FF
SS
DD
RR
TT
AA
PP
VV
EE
33
LL
AA
EE
SS
34
GG
VV
II
RR
AA
JJ
EE
DD
FF
TT
LL
SS
HH
PP
CC
WW
35
II
KK
EE
MMFF
SS
CC
RR
AA
VV
GG
LL
TT
PP
HH
36 37
FF
IISS
RR
PP
TT
GG
LL
AA
VV
KK
EE
38
FFNN
EE
DD
KK
HH
YY
39
FFSS
RR
JJ
XX
KK
II
NN
MM
TT
40
RRDD
41
KK
GG
MMRR
AA
PP
LL
VV
HH
42
VV
GG
XXEE
CC
NN
MM
KK
AA
RR
HH
JJ
TT
SS
LL
FF
YY
43
TT
KK
LL
WW
SS
EE
YY
RR
MM
CC
JJ
44
TTJJ
YY
WW
HH
KK
CC
VV
MM
AA
SS
RR
PP
LL
GG
45
PP
KK
EE
MM
RR
GG
HH
LL
SS
VV
TT
WW
CC
FF
II
46
PPVV
QQII
YY
GG
LL
MM
TT
SS
RR
JJ
CC
KK
NN
47
II
VV
WW
HH
MM
RR
JJ
TT
GG
CC
LL
NN
KK
SS
YY
48
LL
NN
HH
TT
EE
DD
JJ
KK
SS
RR
PP
VV
AA
GG
FF
WW
49
AA
PPEE
SS
LL
MM
JJ
CC
HH
YY
50
SS
YY
EE
LL
JJ
CC
GGPP
MM
KK
VV
RR
TT
WW
AA
II
HH
FF
NN
DD
XX
51
AA
SS
JJ
II
MM
NN
YY
WW
HH
EE
TT
RR
KK
GG
DD
LL
52
RR
AA
PP
CC
LL
IIKK
53
AACC
MM
XX
II
FFWW
LL
JJVV
RR
YY
GG
SS
TT
PP
KK
HH
NN
54
SS
TT
GG
RR
LL
EE
NN
FFPP
JJVV
II
YY
XX
55
VV
MM
JJ
56 57 58 59 60 61 62
II
AA
LL
JJ
EE
NN
RR
HH
KK
TT
GG
FF
63
DD
TT
KK
VV
RR
HH
FF
EE
AA
NN
LL
YY
WW
JJ
PP
SS
XX
CC
GG
II
MM
64
PPAAEE
65
PP
II
DD
WW
SS
EE
VV
RR
JJ
GG
AA
NN
KK
FF
TT
LL
MM
CC
66
SSLL
XX
KK
II
VV
TT
MM
RR
PP
JJ
NN
WW
67
AA
GG
EE
VV
TT
XX
WW
YYKK
LL
RR
SS
68
KKSS
NN
II
MM
FFWW
LL
XX
RR
JJ
PP
GG
TT
VV
EE
AA
DD
HH
69
KK
DD
II
RR
WW
TT
NN
MM
LL
SS
JJ
GG
VV
AA
YY
CC
FF
XX
EE
70
SS
EE
DDWW
CCTT
GG
XX
YY
FF
JJ
RR
KK
LL
MM
AA
VV
NN
II
71
EE
NN
II
VV
KK
FF
GG
CC
TT
RR
AA
PP
LL
HH
SS
72
AA
GGSS
EE
NN
CC
XX
II
KK
MM
JJ
RR
TT
PP
FF
HH
DD
73
LL
PP
GGKK
EE
NN
WW
CC
TT
II
RR
MM
VV
JJ
SS
AA
74
SS
TT
KK
FF
II
EE
NN
DD
RR
75
PP
VV
NN
LL
MM
JJ
RR
WW
XXEE
76
RRKKGG
TT
CC 0
1
2
3
4
bits
1
AAMMNN
2
EEMM
3
WWRR
4
VV
EE
RRWWMM
KK
YY
NN
5
AAII
LL
RR
TT
6
HH
MM
DD
PP
LL
AA
GG
TT
RR
JJ
7
GG
SSKKRR
TT
AA
WW
II
VV
89
VV
HH
GG
10 11
CCSS
LL
GG
EE
WW
DD
12
PPSS
VV
GG
WW
II
MM
RR
NN
KK
TT
DD
AA
FF
13
VVGG
EE
14
WWJJ
15 16
MM
NN
KK
TT
VV
PP
DD
RR
AA
XX
WW
CC
SS
HH
LL
GG
JJ
YY
FF
17
EETTDD
RR
LL
MM
WW
II
FF
PP
AA
18XX
PP
19 20 21
CC
WW
TT
VV
GG
JJ
NN
DD
KK
XX
PP
MM
YY
FF
II
SS
LL
AA
HH
22
CCSS
HH
II
YY
MM
RR
DD
23
SS
RREE
24
LL
25
VVSSYY
26
CC
HH
SSJJ
27
WWXX
28
LLGG
29SS
30
VVMM
RR
KK
XX
GG
31
HH
YY
AA
SS
PP
LL
TT
KK
VV
NN
32
LLTT
SS
GG
RR
PP
VV
AA
EE
33
PP
AA
HH
34
VVRR
SS
PP
TT
LL
WW
35
SS
EE
WW
FFAA
RR
LL
VV
GG
CC
TT
HH
MM
PP
36 37
RR
SS
TT
GG
PP
VV
LL
AA
KK
EE
38
KK
YY
DD
NN
HH
EE
39
SS
RR
II
KK
TT
XX
MM
JJ
NN
40 41
MM
VVRR
HH
AA
LL
PP
42
WW
HHVV
TTLL
SS
RR
NN
CC
JJ
YY
FF
43
LLQQ
JJ
44
MMSS
LL
JJ
RR
PP
VV
GG
45
LL
MM
SS
WW
II
CC
46
LL
PP
KK
TT
SS
RR
CC
NN
47
TTVV
GG
HH
LL
SS
KK
YY
NN
CC
48
RR
PP
GG
VV
AA
JJ
WW
FF
49
SS
LL
JJHH
YY
50
EE
LLSS
GG
PP
HH
TT
CC
VV
RR
AA
DD
NN
KK
XX
MM
FF
II
WW
51
EEGGDD
LL
52
RRTT
53
VVLL
RR
PP
TT
SS
KK
YY
54
XX
TT
RRVV
II
YY
55
RR
CC
WWTT
MM
56 57 58 59 60 61 62
RREETT
VV
GG
JJ
63
VV
DD
EE
HH
GG
RR
NN
KK
FF
MM
YY
II
CC
TT
SS
WW
XX
LL
AA
PP
64 65
RR
TT
AA
GG
LL
MM
KK
JJ
XX
CC
FF
66
NN
RR
XX
VVPP
WW
67
LL
PP
EE
CCMM
TT
GG
SS
RR
68
RRLLXXPP
VV
GG
TT
HH
EE
AA
DD
69
PPTT
SS
LL
MM
AA
EE
CC
FF
70
TTWW
PPAA
MMVV
II
71
MM
AA
HH
TT
RR
PP
GG
LL
SS
72
RRLL
PP
MM
HH
FF
DD
73
LLAA
SS
74
MMRR
75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
HHQQ
NN
23
LL
EE
TT
4
RR
JJ
TTNN
WW
KK
MM
YY
5
LL
TT
FF
KK
II
6
NN
VV
MM
LL
PP
TT
RR
AA
DD
GG
7
RR
KK
AA
JJ
YY
VV
WW
II
89
10 11
NN
LL
GG
DD
EE
WW
12
JJ
SSVV
AA
RR
TT
FF
GG
II
DD
KK
WW
MM
NN
13
RR
GG
EE
14 15 16 17
HH
EE
NN
TT
RR
DD
FF
LL
VV
PP
II
WW
AA
18
JJXX
PP
19 20 21
E
J
Q
H
N
LL
T
F
II
C
W
A
G
K
D
Y
S
P
M
V
X
22
VV
MM
JJ
RR
SS
HH
DD
YY
II
23
RR
DDEE
24
RR
LLJJ
25
SSYY
26SS
27
JJXX
28
LL
DD
29 30
SS
RR
KK
JJ
WW
GG
XX
31
AA
VV
SS
YY
TT
GG
PP
LL
FF
KK
NN
HH
32
LLGG
FF
DD
RR
NN
SS
TT
PP
EE
VV
AA
33 34
R
S
T
F
H
DD
KK
CC
PP
WW
35
NN
RR
VV
AA
GG
LL
CC
HH
TT
PP
36 37
SSPP
GG
LL
RR
TT
VV
AA
KK
EE
38
KK
HH
DD
EE
YY
NN
39
SS
RR
TT
XX
MM
KK
NN
JJ
II
40 41
RR
AA
PP
LL
VV
HH
42
AA
EE
RR
NN
TT
JJ
LL
SS
CC
HH
FF
YY
43
RR
JJ
44
VV
RR
SS
PP
LL
GG
45
GG
LL
TT
VV
EE
SS
WW
FF
II
CC
46
LL
JJ
RR
SS
MM
KK
CC
NN
47
JJ
HH
EE
GG
KK
MM
CC
NN
LL
YY
SS
48
JJ
LL
PP
RR
VV
AA
GG
FF
WW
49
JJ
SS
EE
LL
CC
HH
YY
50 51
RR
TT
EE
NN
GG
DD
LL
52 53
RR
JJ
FF
GG
LL
HH
TT
PP
YY
NN
KK
54
PP
RR
JJ
VV
XX
II
YY
55 56 57 58 59 60 61 62
LLTT
JJ
GG
63 64EE
65
RR
AA
GG
DD
LL
TT
CC
66
RR
TT
LL
KK
PP
JJ
WW
NN
67
GG
JJ
LL
WW
RR
SS
XX
68
FF
RR
EE
NN
LL
MM
PP
GG
DD
VV
TT
HH
AA
69
LL
TT
SS
GG
VV
AA
EE
FF
CC
70
RR
SS
AA
JJ
FF
LL
VV
YY
MM
NN
II
71
AA
GG
RR
TT
LL
PP
HH
SS
72
SS
TT
RR
PP
FF
HH
73
RR
JJ
VV
SS
AA
74RR
75
RR
LL
WWEE
76
QQTT
CC
GG 0
1
2
3
4
bits
1
GG
TT
FF
JJ
MM
HH
NN
2
RRFF
HH
CC
3
GG
VV
LL
TT
EE
4
JJ
WW
NN
MM
KK
YY
5
VV
TT
EE
KK
WW
RRII
6
FF
VV
MM
WW
EE
NN
LL
GG
RR
AA
PP
TT
DD
7
GG
AA
KK
VV
WW
JJ
II
89
10
HH
11
RR
LL
GG
EE
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WW
12
SS
JJ
WW
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AA
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TT
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MM
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NN
13
RR
WW
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GG
EE
14 15 16
PPKK
JJEE
VV
RR
DD
HH
CC
FF
XX
GG
NN
AA
TT
LL
WW
MM
YY
SS
17
SS
JJ
RR
KK
TT
FF
MM
VV
DD
LL
WW
II
PP
AA
18
VVPP
XX
19 20 21 22
TT
CC
EE
VV
SSDD
HH
YY
RR
MM
II
JJ
23
DD
RR
EE
24
RR
VV
LL
KK
YY
25
SS
FF
NN
YY
26
KKSS
27
VV
EE
WW
QQXX
28
RR
GG
VV
KK
AA
29
VV
LL
30
LL
TT
SS
PP
VV
AA
JJ
MM
CC
RR
WW
GG
XX
31
WW
GG
SS
RR
DD
AA
PP
EE
VV
TT
FF
LL
KK
HH
NN
32
LL
WW
SS
YY
FF
RR
TT
VV
AA
PP
EE
33
VV
FF
34
RR
GG
VV
TT
FF
PP
LL
CC
WW
35
FF
RR
SS
EE
AA
GG
LL
VV
TT
PP
HH
36 37
SS
GG
RR
PP
VV
LL
AA
TT
KK
EE
38
DD
EE
HH
KK
YY
NN
39
SS
RR
II
KK
JJ
TT
XX
NN
MM
40 41
GG
RR
PP
VV
AA
LL
HH
42
VV
KK
EE
AA
MM
NN
RR
LL
TT
FF
HH
YY
JJ
SS
43
GG
RR
CC
JJ
44
TT
KK
JJ
MM
RR
WW
PP
VV
HH
SS
LL
GG
45
LL
SS
GG
TT
WW
FF
CC
II
46
GG
WW
LL
TT
YY
RR
SS
KK
CC
NN
47
TT
RR
GG
VV
WW
EE
HH
LL
YY
NN
KK
SS
CC
48
SS
LL
RR
PP
VV
AA
GG
WW
FF
JJ
49
SS
RR
KK
LL
CC
JJ
YY
HH
50 51
SS
VV
RR
TT
WW
EE
GG
YY
KK
DD
LL
52
VVKK
53
SS
VV
RR
MM
WW
CC
NN
TT
PP
LL
GG
HH
KK
54
RR
NN
VV
XX
YY
II
55 56 57 58 59 60 61 62
LL
KK
RR
TT
HH
YY
NN
GG
FF
63 64
AAEE
65
SS
GG
NN
YY
LL
MM
TT
CC
FF
66
LLGG
FF
SS
VV
RR
MM
YY
TT
KK
PP
NN
JJ
WW
67
GG
VV
LL
SS
RR
XX
68
RR
LL
SS
JJ
MM
FF
GG
PP
VV
EE
TT
DD
HH
AA
69
SS
WW
VV
GG
EE
AA
CC
FF
70
TT
KK
GG
JJ
AA
RR
LL
DD
VV
MM
NN
II
71
FF
JJ
GG
MM
RR
WW
CC
AA
VV
TT
LL
HH
PP
SS
72
GG
TTJJ
MM
RR
PP
HH
FF
DD
73
RR
MM
VV
JJ
SS
AA
74
WW
RR
75
RR
WW
LL
YYEE
76
VV
EE
WW
QQGG
TT
CC 0
1
2
3
4
bits
1
JJ
DDQQ
NN
2HH
3
GG
VV
EE
LL
MM
DD
JJ
TT
4
AA
FF
RR
DD
CC
HH
WW
NN
KK
YY
5
AA
KK
LL
FF
HH
EE
TT
NN
WW
RR
DD
II
6
VV
KK
MM
NN
LL
EE
HH
CC
DD
TT
AA
RR
GG
PP
7
SS
KK
EE
AA
MM
JJ
VV
WW
II
89
10 11
RR
PP
LL
HH
GG
DD
EE
WW
12
PP
SS
KK
JJ
TT
AA
GG
WW
VV
RR
II
MM
NN
FF
DD
13
LL
SS
AAWW
DD
EE
GG
14 15 16
PP
II
EE
MM
KK
TT
XX
NN
DDHH
YY
CC
VV
AA
FF
JJ
LL
GG
RR
SS
WW
17
SS
KK
EE
TT
QQMM
RR
LL
DD
VV
PP
FF
WW
AA
II
18
RRJJ
PP
XX
19 20
DD
RR
21 22
GG
KK
EE
CC
VV
SS
MM
RR
HH
DD
II
YY
23
SSRR
DD
CCEE
24
SS
LL
25
SS
FF
NNYY
26
KKTT
SS
27
EE
JJ
XX
28
LLGG
FF
AA
VV
KK
29
LL
GG
30
LL
EE
PP
WW
SS
RR
AA
TT
JJ
VV
KK
GG
XX
31
PP
RR
EE
TT
JJ
FF
YY
SS
AA
LL
VV
HH
KK
NN
32
SS
YY
CC
LL
RR
FF
VV
AA
TT
PP
GG
EE
33
SS
LL
MM
34
GG
RR
YY
LL
SS
TT
EE
PP
FF
HH
CC
MM
WW
35
SS
RR
EE
AA
LL
GG
HH
VV
TT
PP
36 37
SS
RR
TT
GG
LL
VV
AA
KK
PP
EE
38
KK
YY
NN
EE
HH
DD
39
SS
RR
XX
MM
KK
NN
JJ
TT
II
40 41
RR
AA
PP
VV
LL
HH
42
GG
RR
EE
HH
AA
TT
KK
NN
JJ
LL
FF
SS
YY
MM
43
AA
LL
HH
44
MM
JJ
AA
WW
RR
PP
SS
GG
CC
VV
LL
45
PP
EE
GG
LL
MM
YY
SS
JJ
FF
HH
WW
CC
II
46
EE
GG
VV
LL
TT
SS
RR
NN
JJ
KK
CC
MM
47
VV
MM
HH
WW
GG
NN
CC
LL
YY
SS
KK
48
AA
LL
KK
HH
PP
VV
GG
RR
FF
WW
49
KK
MM
LL
CC
JJ
YY
HH
50
S
LL
CC
EE
JJ
AA
GG
MM
NN
KK
PP
TT
RR
FF
WW
HH
II
VV
XX
DD
51
SS
RR
TT
VV
AA
NN
HH
WW
EE
MM
GG
JJ
KK
DD
LL
52
EE
PP
53
LLAA
EE
GG
RR
YY
CC
TT
II
FF
JJ
NN
KK
MM
HH
PP
54
LL
PP
KK
RR
CC
NN
VV
JJ
XX
YY
II
55 56 57 58 59 60 61 62
SS
TT
LL
KK
RR
PP
NN
FF
MM
HH
GG
JJ
63 64
AAEE
65
SS
RR
KK
EE
NN
DD
JJ
AA
MM
TT
GG
LL
FF
CC
66
SS
VV
TT
JJ
RR
LL
NN
PP
WW
67
PP
WW
JJ
RR
VV
GG
LL
SS
68
SS
KK
RR
II
CC
LL
JJ
EE
FF
PP
VV
GG
AA
TT
HH
DD
XX
69
GGKK
RR
DD
WW
CC
TT
SS
LL
VV
MM
EE
FF
AA
YY
70
WW
CC
AA
LL
RR
VV
FF
KK
NN
II
71
KK
AA
CC
VV
TT
RR
GG
LL
HH
PP
SS
72
EE
GG
RR
JJ
PP
DD
HH
FF
MM
73
PP
JJ
WW
VV
AA
SS
74
VVRR
75
RR
VV
LLMM
EE
76
SS
KKTT
GG
CC 0
1
2
3
4
bits
1NN
23
NNTT
CC
4
RRMM
NN
WW
KK
YY
5
KK
VV
RR
LL
EEII
6
VV
LLRR
DD
GG
PP
AA
TT
7
KK
AA
VV
JJ
II
WW
89
10
SS
YY
11
SS
LL
QQDD
GG
WW
EE
12
JJ
SSVV
TT
RR
DD
GG
FF
AA
WW
II
MM
KK
NN
13
RR
GG
EE
14 15 16
PPKK
NN
MM
HH
AA
VV
RR
JJ
LL
SS
CC
YY
DD
TT
GG
FF
WW
17
RR
NN
VV
LL
TT
WW
DD
AA
FF
II
PP
18
RRJJPP
XX
19 20 21
DD
NN
LL
AA
VV
TT
FF
GG
XX
CC
WW
HH
KK
MM
YY
PP
II
SS
JJ
22
AA
VV
SS
JJ
RR
DD
II
HH
YY
23
SS
RR
EE
24
GG
LLJJ
25
RRSS
26
RR
GGSS
27
JJXX
28
GG
SS
DD
VV
29
RRLL
MM
GG
30
SS
RR
TT
JJ
HH
PP
KK
GG
WW
XX
31
DD
RR
TT
VV
GG
PP
LL
AA
KK
NN
32
LLKK
SS
RR
TT
GG
AA
PP
VV
EE
33
VV
SS
34
TT
LL
GG
PP
SS
CC
WW
35
SSRR
MM
LL
AA
TT
GG
VV
HH
PP
36 37
SS
RR
GG
TT
PP
VV
LL
AA
EE
KK
38
HH
YY
DD
KK
NN
EE
39
SS
RR
II
XX
MM
NN
TT
KK
JJ
40 41
VV
RR
LL
AA
PP
HH
42
RR
KK
SS
AA
NN
TT
LL
JJ
FF
HH
YY
43
PP
RRJJ
44
RR
TT
SS
PP
LL
VV
JJ
GG
45
AA
GG
PP
SS
WW
LL
EE
CC
HH
FF
II
46
TT
GG
JJ
CC
YY
LL
RR
SS
NN
KK
MM
47
RR
GG
HH
CC
MM
SS
LL
NN
KK
YY
48
KK
GG
VV
RR
JJ
FF
WW
49
RR
LL
KKJJ
HH
CC
YY
50
LL
KK
JJ
CCVV
RR
PP
GG
FF
DD
AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
JJ
GG
DD
LL
52 53
RR
KK
LL
NN
PP
TT
GG
SS
HH
YY
54
RR
NN
JJ
VV
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
RRTT
KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
FF
EE
70
RR
VV
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
genera
Oscillatoria
Arthrospira
Anabaena
Nostoc
Cyanothece
Spirulina
clades
Pleurocapsa
Prochlorococcus
Gloeobacter
Pseudanabaena
Acaryochloris
Nodosilinea
function logos
Genome
g
tRNA
genes
T
g
C
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g
Nonetheless, tRNA CIFs are Highly
Evolutionarily Conserved
tRNA Class Informative Features
(CIFs) are Grounded in Physics,
not Evolutionary Conservation
tRNA CIFs Predict Functional
Differences Between Human and
Trypanosome aaRSs Rare tRNA Features are
Functionally Informative scores Cyano-MLP
13
0.0
1.0
classification
probabilities0
1
2
3
4
bits
1
AA
SSMM
XX
JJ
FFNN
QQ
2
GG
JJ
CC
WW
YY
HH
3
GG
NN
VV
LL
CC
TT
EE
4
TT
JJ
EE
VV
AA
CC
RR
WW
HH
NN
MM
KK
YY
5
LL
GGKK
QQ
TT
JJ
FF
NN
AA
RR
YY
WW
II
6
QQ
VV
EE
MM
YY
JJ
KK
NN
HH
LL
RR
GG
AA
PP
DD
TT
7
SS
RR
GGNN
YY
KK
AA
VV
WW
JJ
II
89
GG
JJ
FF
10
SS
RRYY
11
RR
AA
NN
LL
TT
PP
QQ
YY
HHDD
EE
GG
WW
12
PP
JJSSWW
KK
VV
GG
MM
TT
NN
RR
AA
II
FF
DD
13
KK
YY
JJ
LL
CC
TT
AA
RR
QQ
MM
WW
GG
EE
14
SS
AA
YY
15
KK
QQJJ
16
PP
II
EE
KK
XX
HH
QQ
RR
SSNN
JJ
DD
WW
YY
VV
AA
TT
GG
FF
CC
MM
LL
17
SS
YY
HH
EE
QQ
NN
KK
TT
VV
RR
DD
FF
LL
WW
PP
AA
II
18
SS
TT
EEXX
PP
19 20 21
EE
JJ
YYLLDD
WW
KK
AA
TT
SS
MM
QQ
HH
XX
NN
FF
GG
PP
II
CC
VV
22
GG
PP
FFWW
NN
XX
CC
KK
EE
JJ
VV
SS
MM
HH
YY
DD
RR
II
23
PP
DD
NN
SS
RREE
24
YY
MM
JJ
LL
KK
25
KK
NN
FF
SS
QQYY
26
TTYY
KK
SS
27
MM
QQ
YYXX
28
SS
QQ
JJ
FF
EE
MM
VV
LL
AA
YY
TT
WW
KK
GG
RR
29
RR
LL
MM
NN
GG
WW
30
PP
VV
SS
LL
QQ
YY
JJ
FF
EE
WW
AA
MM
NNKK
TT
RR
CC
GG
XX
31
RRYY
WW
QQ
JJ
CC
EE
PP
TT
LL
DD
SS
GG
VV
FF
KK
NN
AA
HH
32
GGJJ
YY
DD
LL
MM
KK
SS
FF
TT
RR
AA
QQ
VV
PP
EE
33
AASS
34
VV
SS
JJ
EE
AA
MM
NN
TT
RR
DD
LL
GG
YY
XX
PP
FF
QQ
HH
CC
WW
35
KK
YYNN
FF
EE
MM
SS
RR
AA
GG
LL
QQ
VV
TT
HH
PP
36 37
SS
LL
TT
GG
PP
VV
RR
AA
KK
QQ
EE
38
NN
QQ
EE
HH
DD
YY
KK
39
SS
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XX
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40AA
41
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RR
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42
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47
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48
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49
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53
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54
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55
GG
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56 57 58 59YY
60 61 62
AA
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64
VVYYEE
65
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66
AA
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67
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68
SS
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69
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70
KK
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71
YY
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72
SS
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73
RRTT
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74
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75
TT
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RR
MM
LL
YY
XX
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76
SS
KK
QQ
JJ
MM
EE
YYGG
TT
CC 0
1
2
3
4
bits
1
RR
PP
SS
LL
KK
EE
FF
MM
JJ
XXNN
QQ
2
SS
PPJJ
II
MM
WW
CC
HH
3
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4
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5
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6
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13
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14 15
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16
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17
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QQ
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RR
MM
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II
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18
TT
II
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YYMMPP
XX
19 20 21
EE
LL
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22
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23
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24
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25
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26
VV
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27
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28
PP
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DD
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29
RR
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30
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31
MM
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32
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33
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34
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35
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36 37
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38
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39
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40DD
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42
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43
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44
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45
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46
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47
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48
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XX
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49
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50
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53
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54
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55
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56
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57 58 59
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60 61 62
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63
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WW
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64
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65
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66
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67
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68
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69
KK
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70
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71
GG
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72
LL
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73
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74
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75
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76
RR
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XX
EETT
GG
CC 0
1
2
3
4
bits
1
AA
GG
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KKNN
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23
LL
RR
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FF
EE
CC
TT
4
AA
GG
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6
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89
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12
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13
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14 15 16
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17
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18
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19 20 21
R
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22
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23
PP
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24
LL
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25
TT
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26
PP
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MMSS
27
AA
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28
SS
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VV
29
RR
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NN
GG
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30
PP
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FF
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SS
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XX
31
EE
XX
RR
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CC
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AA
PP
LL
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NN
32
LLGG
II
KK
NN
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AA
PP
VV
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33
LL
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34
GG
VV
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HH
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CC
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35
II
KK
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AA
VV
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LL
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QQ
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HH
36 37
FF
IISS
RR
PP
TT
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LL
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VV
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38
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HH
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39
FFSS
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40
RRDD
41
KK
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PP
LL
VV
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42
VV
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43
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44
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45
PP
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WW
CC
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II
46
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YY
GG
LL
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TT
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NN
47
II
VV
WW
HH
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NN
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48
LL
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AA
GG
FF
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49
AA
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LL
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50
SS
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LL
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CC
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QQ
TT
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HH
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XX
51
AA
SS
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II
MM
NN
QQ
YY
WW
HH
EE
TT
RR
KK
GG
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LL
52
RR
AA
PP
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53
AACC
MM
XX
II
FFWW
LL
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RR
YY
GG
SS
TT
PP
KK
HH
NN
QQ
54
SS
TT
GG
RR
LL
EE
NN
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II
YY
XX
55
VV
MM
JJ
56 57 58 59 60 61 62
II
AA
LL
JJ
EE
NN
RR
QQ
HH
KK
TT
GG
FF
63
DD
TT
KK
VV
RR
HH
FF
EE
AA
QQ
NN
LL
YY
WW
JJ
PP
SS
XX
CC
GG
II
MM
64
PPAAEE
65
PP
II
DD
WW
SS
EE
VV
RR
JJ
GG
AA
NN
KK
FF
TT
LL
MM
CC
66
SSLL
XX
KK
II
VV
TT
MM
RR
PP
JJ
NN
WW
QQ
67
AA
GG
EE
VV
TT
XX
QQ
WW
YYKK
LL
RR
SS
68
KKSS
NN
II
MM
FFWW
LL
XX
RR
JJ
PP
GG
TT
VV
EE
AA
DD
HH
69
KK
DD
II
RR
WW
TT
NN
MM
LL
SS
JJ
GG
VV
AA
YY
CC
FF
XX
EE
70
SS
EE
DDWW
CCTT
GG
XX
YY
FF
JJ
RR
QQ
KK
LL
MM
AA
VV
NN
II
71
EE
NN
II
VV
KK
QQ
FF
GG
CC
TT
RR
AA
PP
LL
HH
SS
72
AA
GGSS
EE
NN
CC
XX
II
KK
MM
JJ
RR
TT
PP
FF
HH
DD
73
LL
PP
GGKK
EE
NN
WW
CC
TT
II
RR
MM
VV
JJ
SS
AA
74
SS
TT
KK
FF
II
EE
NN
DD
RR
75
PP
VV
NN
LL
MM
JJ
RR
WW
XXEE
76
RRKKGG
TT
CC 0
1
2
3
4
bits
1
AAMMNN
QQ
2
EEMM
3
WWRR
4
VV
EE
RRWWMM
KK
YY
NN
5
AAII
LL
RR
TT
6
HH
MM
DD
PP
LL
AA
GG
TT
RR
JJ
7
GG
SSKKRR
TT
AA
WW
II
VV
89
VV
HH
GG
10 11
CCSS
LL
GG
EE
WW
DD
12
PPSS
VV
GG
WW
II
MM
RR
NN
KK
TT
DD
AA
FF
13
VVGG
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14
WWJJ
15 16
MM
NN
KK
TT
VV
PP
DD
RR
AA
QQ
XX
WW
CC
SS
HH
LL
GG
JJ
YY
FF
17
EETTDD
RR
LL
MM
WW
II
FF
PP
AA
18XX
PP
19 20 21
CC
WW
TT
VV
GG
QQ
JJ
NN
DD
KK
XX
PP
MM
YY
FF
II
SS
LL
AA
HH
22
CCSS
HH
II
YY
MM
RR
DD
23
SS
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24
LL
25
VVSSYY
26
CC
HH
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27
WWXX
28
LLGG
29SS
30
VVMM
QQ
RR
KK
XX
GG
31
HH
YY
AA
SS
PP
LL
TT
KK
VV
NN
32
LLTT
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GG
RR
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VV
AA
EE
33
PP
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34
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35
SS
EE
WW
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CC
TT
HH
QQ
MM
PP
36 37
RR
SS
TT
GG
PP
VV
LL
AA
KK
QQ
EE
38
KK
QQ
YY
DD
NN
HH
EE
39
SS
RR
II
KK
TT
XX
MM
JJ
NN
40 41
MM
VVRR
HH
AA
LL
PP
QQ
42
WW
HHVV
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SS
RR
NN
CC
JJ
YY
FF
43
LLQQ
JJ
44
MMSS
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PP
VV
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45
LL
MM
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CC
46
LL
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NN
47
TTVV
GG
HH
LL
SS
KK
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NN
CC
48
RR
PP
GG
VV
AA
JJ
WW
FF
49
SS
LL
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YY
QQ
50
EE
LLSS
GG
PP
HH
TT
CC
VV
RR
AA
QQ
DD
NN
KK
XX
MM
FF
II
WW
51
EEGGDD
LL
52
RRTT
53
VVLL
RR
PP
TT
SS
KK
QQ
YY
54
XX
TT
RRVV
II
YY
55
RR
CC
WWTT
MM
56 57 58 59 60 61 62
RREETT
VV
GG
JJ
63
VV
DD
EE
HH
GG
QQ
RR
NN
KK
FF
MM
YY
II
CC
TT
SS
WW
XX
LL
AA
PP
64 65
RR
TT
AA
GG
LL
MM
KK
JJ
XX
CC
FF
66
NN
RR
XX
VVPP
WW
QQ
67
LL
PP
EE
CCMM
TT
GG
SS
RR
68
RRLLXXPP
VV
GG
TT
HH
EE
AA
DD
69
PPTT
SS
LL
MM
AA
EE
CC
FF
70
TTWW
PPAA
MMVV
II
71
MM
AA
HH
TT
RR
PP
GG
LL
SS
72
RRLL
PP
MM
HH
FF
DD
73
LLAA
SS
74
MMRR
75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
HHQQ
NN
23
LL
EE
TT
4
RR
JJ
TTNN
WW
KK
MM
YY
5
LL
TT
FF
KK
II
6
NN
VV
MM
LL
PP
TT
RR
QQ
AA
DD
GG
7
RR
KK
AA
JJ
YY
VV
WW
II
89
10 11
QQ
NN
LL
GG
DD
EE
WW
12
JJ
SSVV
AA
RR
TT
FF
GG
II
DD
KK
WW
MM
NN
13
RR
QQ
GG
EE
14 15 16 17
HH
EE
NN
TT
QQ
RR
DD
FF
LL
VV
PP
II
WW
AA
18
JJXX
PP
19 20 21
E
J
Q
H
N
LL
T
F
II
C
W
A
G
K
D
Y
S
P
M
V
X
22
VV
MM
JJ
RR
SS
HH
DD
YY
II
23
RR
DDEE
24
RR
LLJJ
25
SSYY
26SS
27
QQ
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28
LL
QQ
DD
29 30
SS
QQ
RR
KK
JJ
WW
GG
XX
31
AA
VV
SS
YY
TT
GG
PP
LL
FF
KK
NN
HH
32
LLGG
FF
QQ
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RR
NN
SS
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EE
VV
AA
33 34
R
S
T
F
H
DD
KK
CC
PP
WW
35
NN
RR
VV
QQ
AA
GG
LL
CC
HH
TT
PP
36 37
SSPP
GG
LL
RR
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38
KK
HH
DD
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QQ
39
SS
RR
TT
XX
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II
40 41
RR
AA
PP
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VV
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42
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43
RR
QQ
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44
VV
RR
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45
GG
LL
TT
VV
EE
SS
WW
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II
CC
46
LL
JJ
QQ
RR
SS
MM
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CC
NN
47
JJ
HH
EE
GG
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MM
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NN
LL
YY
SS
48
JJ
LL
PP
RR
VV
AA
GG
FF
WW
49
JJ
SS
EE
LL
CC
QQ
HH
YY
50 51
RR
TT
EE
NN
GG
QQ
DD
LL
52 53
RR
JJ
FF
GG
LL
HH
TT
PP
YY
NN
KK
QQ
54
PP
RR
JJ
VV
XX
II
YY
55 56 57 58 59 60 61 62
LLTT
JJ
GG
63 64EE
65
RR
AA
GG
DD
LL
TT
CC
66
RR
TT
LL
KK
PP
JJ
WW
QQ
NN
67
GG
JJ
LL
WW
RR
SS
XX
68
FF
RR
EE
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TT
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69
LL
TT
SS
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VV
AA
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70
RR
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71
AA
GG
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72
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PP
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73
RR
JJ
VV
SS
AA
74RR
75
RR
LL
WWEE
76
QQTT
CC
GG 0
1
2
3
4
bits
1
GG
TT
FF
JJ
MM
HH
QQ
NN
2
RRFF
HH
CC
3
GG
VV
QQ
LL
TT
EE
4
JJ
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MM
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5
VV
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6
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VV
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QQ
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AA
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7
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AA
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89
10
HH
11
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LL
QQ
GG
EE
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WW
12
SS
JJ
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GG
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II
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NN
13
QQ
RR
WW
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GG
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14 15 16
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QQ
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17
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18
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19 20 21 22
TT
CC
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HH
YY
RR
MM
II
JJ
23
DD
RR
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24
RR
VV
LL
KK
YY
25
SS
FF
NN
YY
26
KKSS
27
VV
EE
WW
QQXX
28
RR
GG
VV
KK
QQ
AA
29
VV
LL
QQ
30
LL
TT
SS
PP
VV
AA
JJ
MM
QQ
CC
RR
WW
GG
XX
31
WW
GG
SS
RR
DD
AA
PP
EE
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FF
LL
KK
HH
NN
32
LL
WW
SS
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TT
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AA
PP
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33
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FF
34
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GG
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WW
35
FF
RR
SS
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AA
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GG
LL
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36 37
SS
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AA
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38
DD
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39
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40 41
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RR
PP
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42
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44
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45
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46
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SS
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CC
NN
47
TT
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GG
VV
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48
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LL
RR
PP
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AA
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49
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RR
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CC
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QQ
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50 51
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52
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53
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VV
RR
MM
WW
CC
NN
TT
PP
LL
QQ
GG
HH
KK
54
RR
NN
VV
XX
YY
II
55 56 57 58 59 60 61 62
LL
KK
RR
TT
HH
YY
NN
GG
FF
63 64
AAEE
65
SS
GG
NN
YY
LL
MM
TT
CC
FF
66
LLGG
FF
SS
VV
RR
MM
YY
TT
KK
PP
NN
JJ
WW
QQ
67
GG
VV
LL
QQ
SS
RR
XX
68
RR
LL
SS
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MM
FF
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PP
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HH
AA
69
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WW
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70
TT
KK
GG
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AA
QQ
RR
LL
DD
VV
MM
NN
II
71
FF
JJ
GG
MM
RR
WW
CC
AA
VV
TT
LL
HH
PP
SS
72
GG
TTJJ
MM
RR
PP
HH
FF
DD
73
RR
MM
VV
JJ
SS
AA
74
WW
RR
75
RR
WW
LL
YYEE
76
VV
EE
WW
QQGG
TT
CC 0
1
2
3
4
bits
1
JJ
DDQQ
NN
2HH
3
GG
VV
EE
LL
MM
DD
JJ
TT
4
AA
FF
RR
DD
CC
HH
WW
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KK
YY
5
AA
KK
LL
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HH
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WW
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DD
II
6
VV
KK
MM
NN
LL
QQ
EE
HH
CC
DD
TT
AA
RR
GG
PP
7
SS
KK
EE
AA
MM
JJ
VV
WW
II
89
10 11
RR
PP
LL
HH
GG
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EE
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12
PP
SS
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RR
II
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13
LL
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14 15 16
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II
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XX
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YY
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VV
AA
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LL
GG
RR
SS
WW
17
SS
KK
EE
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RR
LL
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VV
PP
FF
WW
AA
II
18
RRJJ
PP
XX
19 20
DD
RR
21 22
GG
KK
EE
CC
VV
SS
MM
RR
HH
DD
II
YY
23
SSRR
DD
CCEE
24
SS
LL
25
SS
FF
NNYY
26
KKTT
SS
27
QQ
EE
JJ
XX
28
LLGG
FF
AA
VV
KK
29
LL
GG
30
LL
EE
PP
QQ
WW
SS
RR
AA
TT
JJ
VV
KK
GG
XX
31
PP
RR
EE
TT
JJ
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AA
LL
VV
HH
KK
NN
32
SS
YY
CC
LL
RR
FF
QQ
VV
AA
TT
PP
GG
EE
33
SS
LL
MM
34
GG
RR
QQ
YY
LL
SS
TT
EE
PP
FF
HH
CC
MM
WW
35
SS
RR
EE
AA
LL
GG
QQ
HH
VV
TT
PP
36 37
SS
RR
TT
GG
LL
VV
AA
KK
PP
QQ
EE
38
KK
YY
NN
EE
QQ
HH
DD
39
SS
RR
XX
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TT
II
40 41
RR
AA
PP
VV
LL
QQ
HH
42
GG
RR
EE
HH
AA
TT
KK
NN
JJ
LL
FF
SS
YY
MM
43
AA
LL
HH
44
QQ
MM
JJ
AA
WW
RR
PP
SS
GG
CC
VV
LL
45
PP
EE
GG
LL
MM
YY
SS
JJ
FF
HH
WW
CC
II
46
EE
GG
VV
LL
TT
SS
RR
NN
JJ
KK
CC
MM
47
VV
MM
HH
WW
QQ
GG
NN
CC
LL
YY
SS
KK
48
AA
LL
KK
HH
PP
VV
GG
RR
FF
WW
49
KK
MM
LL
CC
JJ
YY
HH
QQ
50
S
LL
CC
EE
JJ
AA
GG
QQ
MM
NN
KK
PP
TT
RR
FF
WW
HH
II
VV
XX
DD
51
SS
RR
TT
VV
AA
NN
HH
WW
EE
QQ
MM
GG
JJ
KK
DD
LL
52
EE
PP
53
LLAA
EE
GG
QQ
RR
YY
CC
TT
II
FF
JJ
NN
KK
MM
HH
PP
54
LL
PP
KK
RR
CC
NN
VV
JJ
XX
YY
II
55 56 57 58 59 60 61 62
SS
TT
LL
KK
RR
PP
NN
QQ
FF
MM
HH
GG
JJ
63 64
AAEE
65
SS
RR
KK
EE
NN
DD
JJ
AA
MM
TT
GG
LL
FF
CC
66
SS
VV
TT
JJ
RR
LL
NN
PP
WW
QQ
67
PP
WW
JJ
RR
VV
GG
LL
SS
QQ
68
SS
KK
RR
II
CC
LL
JJ
EE
FF
PP
VV
GG
AA
TT
HH
DD
XX
69
GGKK
RR
DD
WW
CC
TT
SS
LL
VV
MM
EE
FF
AA
YY
70
WW
CC
AA
LL
RR
QQ
VV
FF
KK
NN
II
71
KK
QQ
AA
CC
VV
TT
RR
GG
LL
HH
PP
SS
72
EE
GG
RR
JJ
PP
DD
HH
FF
MM
73
PP
JJ
WW
VV
AA
SS
74
VVRR
75
RR
VV
LLMM
EE
76
SS
KKTT
GG
CC 0
1
2
3
4
bits
1NN
QQ
23
NNTT
CC
4
RRMM
NN
WW
KK
YY
5
KK
VV
RR
LL
EEII
6
VV
LLRR
DD
GG
PP
AA
TT
7
KK
AA
VV
JJ
II
WW
89
10
SS
YY
11
SS
LL
QQDD
GG
WW
EE
12
JJ
SSVV
TT
RR
DD
GG
FF
AA
WW
II
MM
KK
NN
13
RR
QQ
GG
EE
14 15 16
PPKK
NN
MM
HH
QQ
AA
VV
RR
JJ
LL
SS
CC
YY
DD
TT
GG
FF
WW
17
RR
QQ
NN
VV
LL
TT
WW
DD
AA
FF
II
PP
18
RRJJPP
XX
19 20 21
DD
QQ
NN
LL
AA
VV
TT
FF
GG
XX
CC
WW
HH
KK
MM
YY
PP
II
SS
JJ
22
AA
VV
SS
JJ
RR
DD
II
HH
YY
23
SS
RR
EE
24
GG
LLJJ
25
RRSS
26
RR
GGSS
27
JJXX
28
GG
SS
DD
VV
29
RRLL
MM
GG
30
SS
RR
TT
QQ
JJ
HH
PP
KK
GG
WW
XX
31
DD
RR
TT
VV
GG
PP
LL
AA
KK
NN
32
LLKK
QQ
SS
RR
TT
GG
AA
PP
VV
EE
33
VV
SS
34
TT
LL
GG
PP
SS
CC
WW
35
SSRR
MM
LL
AA
TT
GG
VV
HH
QQ
PP
36 37
SS
RR
GG
TT
PP
VV
LL
AA
EE
QQ
KK
38
HH
YY
QQ
DD
KK
NN
EE
39
SS
RR
II
XX
MM
NN
TT
KK
JJ
40 41
VV
RR
LL
AA
PP
HH
QQ
42
RR
KK
SS
AA
NN
TT
LL
JJ
FF
HH
YY
43
PP
RRJJ
44
RR
TT
SS
QQ
PP
LL
VV
JJ
GG
45
AA
GG
PP
SS
WW
LL
EE
CC
HH
FF
II
46
TT
GG
JJ
CC
YY
LL
RR
SS
NN
KK
MM
47
RR
GG
HH
CC
MM
SS
LL
NN
KK
YY
48
KK
GG
VV
RR
JJ
FF
WW
49
RR
LL
KKJJ
QQ
HH
CC
YY
50
LL
KK
JJ
CCVV
RR
QQ
PP
GG
FF
DD
AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
QQ
JJ
GG
DD
LL
52 53
RR
KK
LL
QQ
NN
PP
TT
GG
SS
HH
YY
54
RR
NN
JJ
VV
QQ
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
RRTT
KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
QQ
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
QQ
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
QQ
FF
EE
70
RR
VV
QQ
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
function logos
Genome
g
tRNA
genes
T
g
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g tRNA CIFs and Eukaryotic tRNA
Gene Clusters are Conserved tRNA Class Informative Features
(CIFs) are Grounded in Physics,
not Evolutionary Conservation
Outline
Differences Between Human and
Trypanosome aaRSs Rare tRNA Features are
Functionally Informative scores Cyano-MLP
13
0.0
1.0
classification
probabilities0
1
2
3
4
bits
1
AA
SSMM
XX
JJ
FFNN
2
GG
JJ
CC
WW
YY
HH
3
GG
NN
VV
LL
CC
TT
EE
4
TT
JJ
EE
VV
AA
CC
RR
WW
HH
NN
MM
KK
YY
5
LL
GGKK
TT
JJ
FF
NN
AA
RR
YY
WW
II
6
VV
EE
MM
YY
JJ
KK
NN
HH
LL
RR
GG
AA
PP
DD
TT
7
SS
RR
GGNN
YY
KK
AA
VV
WW
JJ
II
89
GG
JJ
FF
10
SS
RRYY
11
RR
AA
NN
LL
TT
PP
YY
HHDD
EE
GG
WW
12
PP
JJSSWW
KK
VV
GG
MM
TT
NN
RR
AA
II
FF
DD
13
KK
YY
JJ
LL
CC
TT
AA
RR
MM
WW
GG
EE
14
SS
AA
YY
15
KK
QQJJ
16
PP
II
EE
KK
XX
HH
RR
SSNN
JJ
DD
WW
YY
VV
AA
TT
GG
FF
CC
MM
LL
17
SS
YY
HH
EE
NN
KK
TT
VV
RR
DD
FF
LL
WW
PP
AA
II
18
SS
TT
EEXX
PP
19 20 21
EE
JJ
YYLLDD
WW
KK
AA
TT
SS
MM
HH
XX
NN
FF
GG
PP
II
CC
VV
22
GG
PP
FFWW
NN
XX
CC
KK
EE
JJ
VV
SS
MM
HH
YY
DD
RR
II
23
PP
DD
NN
SS
RREE
24
YY
MM
JJ
LL
KK
25
KK
NN
FF
SS
QQYY
26
TTYY
KK
SS
27
MM
YYXX
28
SS
JJ
FF
EE
MM
VV
LL
AA
YY
TT
WW
KK
GG
RR
29
RR
LL
MM
NN
GG
WW
30
PP
VV
SS
LL
YY
JJ
FF
EE
WW
AA
MM
NNKK
TT
RR
CC
GG
XX
31
RRYY
WW
JJ
CC
EE
PP
TT
LL
DD
SS
GG
VV
FF
KK
NN
AA
HH
32
GGJJ
YY
DD
LL
MM
KK
SS
FF
TT
RR
AA
VV
PP
EE
33
AASS
34
VV
SS
JJ
EE
AA
MM
NN
TT
RR
DD
LL
GG
YY
XX
PP
FF
HH
CC
WW
35
KK
YYNN
FF
EE
MM
SS
RR
AA
GG
LL
VV
TT
HH
PP
36 37
SS
LL
TT
GG
PP
VV
RR
AA
KK
EE
38
NN
EE
HH
DD
YY
KK
39
SS
RR
KK
TT
XX
MM
II
JJ
NN
40AA
41
SSGG
RR
AA
PP
LL
VV
HH
42
NN
GG
EE
RR
AA
KK
HH
TT
YY
FF
LL
SS
JJ
43
TT
GG
LL
EE
DD
NN
AA
SS
MM
RR
YY
CC
JJ
44
FF
AA
NN
HH
JJ
KK
XX
MM
YY
RR
PP
VV
SS
LL
GG
45
KK
GG
PP
DD
MM
HH
RR
SS
EE
LL
YY
TT
FF
CC
WW
II
46
JJ
FF
MM
GG
YY
RR
WW
TT
CC
LL
SS
KK
NN
47
AAQQ
FF
DDTT
WW
RR
JJ
EE
GG
MM
HH
CC
LL
YY
KK
NN
SS
48
TT
KK
HH
SS
NN
AA
JJ
LL
CC
MM
PP
VV
GG
RR
FF
WW
49
RR
KK
FF
EE
SS
JJ
CC
HH
LL
YY
50
YY
JJ
SS
LL
EE
CC
GGQQMM
WW
AA
KK
RR
FF
TT
NN
VV
PP
XX
II
DD
HH
51
SS
PP
FF
CC
HH
JJ
VV
YY
TT
AA
NN
RR
MM
WW
EE
GG
KK
DD
LL
52
GG
II
KK
53
VV
JJ
AA
CC
MM
LL
KK
GG
RR
WW
YY
TT
SS
FF
PP
HH
NN
54
TT
MM
LL
PP
NN
JJ
VV
XX
YY
II
55
GG
NN
YY
56 57 58 59YY
60 61 62
AA
RRDD
EE
CC
XX
KK
LL
MM
YY
FF
NN
TT
GG
HH
JJ
63
DD
FF
TT
YY
AA
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66
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67
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68
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69
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70
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71
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72
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73
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75
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LL
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76
SS
KK
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CC 0
1
2
3
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bits
1
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2
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3
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14 15
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16
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17
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18
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19 20 21
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26
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28
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29
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31
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32
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35
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36 37
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48
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54
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55
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56
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57 58 59
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60 61 62
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63
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64
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65
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66
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67
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68
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69
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70
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71
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72
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73
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75
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76
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CC 0
1
2
3
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bits
1
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23
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4
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13
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14 15 16
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17
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18
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19 20 21
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23
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24
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25
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26
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27
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28
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29
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30
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31
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32
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33
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34
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35
II
KK
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36 37
FF
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38
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39
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40
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41
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47
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48
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50
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52
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53
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54
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55
VV
MM
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56 57 58 59 60 61 62
II
AA
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63
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64
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65
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66
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67
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68
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69
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70
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71
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72
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73
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74
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75
PP
VV
NN
LL
MM
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76
RRKKGG
TT
CC 0
1
2
3
4
bits
1
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2
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3
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4
VV
EE
RRWWMM
KK
YY
NN
5
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6
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MM
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7
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89
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10 11
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12
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13
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15 16
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17
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18XX
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19 20 21
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22
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23
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24
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25
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26
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27
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29SS
30
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31
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32
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35
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36 37
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39
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40 41
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42
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43
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44
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45
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46
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47
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48
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49
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50
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51
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52
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53
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54
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55
RR
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56 57 58 59 60 61 62
RREETT
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JJ
63
VV
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64 65
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66
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67
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68
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69
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70
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71
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72
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MM
HH
FF
DD
73
LLAA
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74
MMRR
75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
HHQQ
NN
23
LL
EE
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4
RR
JJ
TTNN
WW
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MM
YY
5
LL
TT
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II
6
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89
10 11
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14 15 16 17
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19 20 21
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24
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25
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27
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28
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29 30
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31
AA
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32
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33 34
R
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35
NN
RR
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AA
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36 37
SSPP
GG
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VV
AA
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EE
38
KK
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NN
39
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40 41
RR
AA
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42
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43
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45
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46
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47
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48
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49
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50 51
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52 53
RR
JJ
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PP
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54
PP
RR
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VV
XX
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55 56 57 58 59 60 61 62
LLTT
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GG
63 64EE
65
RR
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66
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NN
67
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68
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69
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70
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71
AA
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HH
SS
72
SS
TT
RR
PP
FF
HH
73
RR
JJ
VV
SS
AA
74RR
75
RR
LL
WWEE
76
QQTT
CC
GG 0
1
2
3
4
bits
1
GG
TT
FF
JJ
MM
HH
NN
2
RRFF
HH
CC
3
GG
VV
LL
TT
EE
4
JJ
WW
NN
MM
KK
YY
5
VV
TT
EE
KK
WW
RRII
6
FF
VV
MM
WW
EE
NN
LL
GG
RR
AA
PP
TT
DD
7
GG
AA
KK
VV
WW
JJ
II
89
10
HH
11
RR
LL
GG
EE
DD
WW
12
SS
JJ
WW
VV
RR
KK
AA
GG
TT
II
MM
FF
DD
NN
13
RR
WW
YY
GG
EE
14 15 16
PPKK
JJEE
VV
RR
DD
HH
CC
FF
XX
GG
NN
AA
TT
LL
WW
MM
YY
SS
17
SS
JJ
RR
KK
TT
FF
MM
VV
DD
LL
WW
II
PP
AA
18
VVPP
XX
19 20 21 22
TT
CC
EE
VV
SSDD
HH
YY
RR
MM
II
JJ
23
DD
RR
EE
24
RR
VV
LL
KK
YY
25
SS
FF
NN
YY
26
KKSS
27
VV
EE
WW
QQXX
28
RR
GG
VV
KK
AA
29
VV
LL
30
LL
TT
SS
PP
VV
AA
JJ
MM
CC
RR
WW
GG
XX
31
WW
GG
SS
RR
DD
AA
PP
EE
VV
TT
FF
LL
KK
HH
NN
32
LL
WW
SS
YY
FF
RR
TT
VV
AA
PP
EE
33
VV
FF
34
RR
GG
VV
TT
FF
PP
LL
CC
WW
35
FF
RR
SS
EE
AA
GG
LL
VV
TT
PP
HH
36 37
SS
GG
RR
PP
VV
LL
AA
TT
KK
EE
38
DD
EE
HH
KK
YY
NN
39
SS
RR
II
KK
JJ
TT
XX
NN
MM
40 41
GG
RR
PP
VV
AA
LL
HH
42
VV
KK
EE
AA
MM
NN
RR
LL
TT
FF
HH
YY
JJ
SS
43
GG
RR
CC
JJ
44
TT
KK
JJ
MM
RR
WW
PP
VV
HH
SS
LL
GG
45
LL
SS
GG
TT
WW
FF
CC
II
46
GG
WW
LL
TT
YY
RR
SS
KK
CC
NN
47
TT
RR
GG
VV
WW
EE
HH
LL
YY
NN
KK
SS
CC
48
SS
LL
RR
PP
VV
AA
GG
WW
FF
JJ
49
SS
RR
KK
LL
CC
JJ
YY
HH
50 51
SS
VV
RR
TT
WW
EE
GG
YY
KK
DD
LL
52
VVKK
53
SS
VV
RR
MM
WW
CC
NN
TT
PP
LL
GG
HH
KK
54
RR
NN
VV
XX
YY
II
55 56 57 58 59 60 61 62
LL
KK
RR
TT
HH
YY
NN
GG
FF
63 64
AAEE
65
SS
GG
NN
YY
LL
MM
TT
CC
FF
66
LLGG
FF
SS
VV
RR
MM
YY
TT
KK
PP
NN
JJ
WW
67
GG
VV
LL
SS
RR
XX
68
RR
LL
SS
JJ
MM
FF
GG
PP
VV
EE
TT
DD
HH
AA
69
SS
WW
VV
GG
EE
AA
CC
FF
70
TT
KK
GG
JJ
AA
RR
LL
DD
VV
MM
NN
II
71
FF
JJ
GG
MM
RR
WW
CC
AA
VV
TT
LL
HH
PP
SS
72
GG
TTJJ
MM
RR
PP
HH
FF
DD
73
RR
MM
VV
JJ
SS
AA
74
WW
RR
75
RR
WW
LL
YYEE
76
VV
EE
WW
QQGG
TT
CC 0
1
2
3
4
bits
1
JJ
DDQQ
NN
2HH
3
GG
VV
EE
LL
MM
DD
JJ
TT
4
AA
FF
RR
DD
CC
HH
WW
NN
KK
YY
5
AA
KK
LL
FF
HH
EE
TT
NN
WW
RR
DD
II
6
VV
KK
MM
NN
LL
EE
HH
CC
DD
TT
AA
RR
GG
PP
7
SS
KK
EE
AA
MM
JJ
VV
WW
II
89
10 11
RR
PP
LL
HH
GG
DD
EE
WW
12
PP
SS
KK
JJ
TT
AA
GG
WW
VV
RR
II
MM
NN
FF
DD
13
LL
SS
AAWW
DD
EE
GG
14 15 16
PP
II
EE
MM
KK
TT
XX
NN
DDHH
YY
CC
VV
AA
FF
JJ
LL
GG
RR
SS
WW
17
SS
KK
EE
TT
QQMM
RR
LL
DD
VV
PP
FF
WW
AA
II
18
RRJJ
PP
XX
19 20
DD
RR
21 22
GG
KK
EE
CC
VV
SS
MM
RR
HH
DD
II
YY
23
SSRR
DD
CCEE
24
SS
LL
25
SS
FF
NNYY
26
KKTT
SS
27
EE
JJ
XX
28
LLGG
FF
AA
VV
KK
29
LL
GG
30
LL
EE
PP
WW
SS
RR
AA
TT
JJ
VV
KK
GG
XX
31
PP
RR
EE
TT
JJ
FF
YY
SS
AA
LL
VV
HH
KK
NN
32
SS
YY
CC
LL
RR
FF
VV
AA
TT
PP
GG
EE
33
SS
LL
MM
34
GG
RR
YY
LL
SS
TT
EE
PP
FF
HH
CC
MM
WW
35
SS
RR
EE
AA
LL
GG
HH
VV
TT
PP
36 37
SS
RR
TT
GG
LL
VV
AA
KK
PP
EE
38
KK
YY
NN
EE
HH
DD
39
SS
RR
XX
MM
KK
NN
JJ
TT
II
40 41
RR
AA
PP
VV
LL
HH
42
GG
RR
EE
HH
AA
TT
KK
NN
JJ
LL
FF
SS
YY
MM
43
AA
LL
HH
44
MM
JJ
AA
WW
RR
PP
SS
GG
CC
VV
LL
45
PP
EE
GG
LL
MM
YY
SS
JJ
FF
HH
WW
CC
II
46
EE
GG
VV
LL
TT
SS
RR
NN
JJ
KK
CC
MM
47
VV
MM
HH
WW
GG
NN
CC
LL
YY
SS
KK
48
AA
LL
KK
HH
PP
VV
GG
RR
FF
WW
49
KK
MM
LL
CC
JJ
YY
HH
50
S
LL
CC
EE
JJ
AA
GG
MM
NN
KK
PP
TT
RR
FF
WW
HH
II
VV
XX
DD
51
SS
RR
TT
VV
AA
NN
HH
WW
EE
MM
GG
JJ
KK
DD
LL
52
EE
PP
53
LLAA
EE
GG
RR
YY
CC
TT
II
FF
JJ
NN
KK
MM
HH
PP
54
LL
PP
KK
RR
CC
NN
VV
JJ
XX
YY
II
55 56 57 58 59 60 61 62
SS
TT
LL
KK
RR
PP
NN
FF
MM
HH
GG
JJ
63 64
AAEE
65
SS
RR
KK
EE
NN
DD
JJ
AA
MM
TT
GG
LL
FF
CC
66
SS
VV
TT
JJ
RR
LL
NN
PP
WW
67
PP
WW
JJ
RR
VV
GG
LL
SS
68
SS
KK
RR
II
CC
LL
JJ
EE
FF
PP
VV
GG
AA
TT
HH
DD
XX
69
GGKK
RR
DD
WW
CC
TT
SS
LL
VV
MM
EE
FF
AA
YY
70
WW
CC
AA
LL
RR
VV
FF
KK
NN
II
71
KK
AA
CC
VV
TT
RR
GG
LL
HH
PP
SS
72
EE
GG
RR
JJ
PP
DD
HH
FF
MM
73
PP
JJ
WW
VV
AA
SS
74
VVRR
75
RR
VV
LLMM
EE
76
SS
KKTT
GG
CC 0
1
2
3
4
bits
1NN
23
NNTT
CC
4
RRMM
NN
WW
KK
YY
5
KK
VV
RR
LL
EEII
6
VV
LLRR
DD
GG
PP
AA
TT
7
KK
AA
VV
JJ
II
WW
89
10
SS
YY
11
SS
LL
QQDD
GG
WW
EE
12
JJ
SSVV
TT
RR
DD
GG
FF
AA
WW
II
MM
KK
NN
13
RR
GG
EE
14 15 16
PPKK
NN
MM
HH
AA
VV
RR
JJ
LL
SS
CC
YY
DD
TT
GG
FF
WW
17
RR
NN
VV
LL
TT
WW
DD
AA
FF
II
PP
18
RRJJPP
XX
19 20 21
DD
NN
LL
AA
VV
TT
FF
GG
XX
CC
WW
HH
KK
MM
YY
PP
II
SS
JJ
22
AA
VV
SS
JJ
RR
DD
II
HH
YY
23
SS
RR
EE
24
GG
LLJJ
25
RRSS
26
RR
GGSS
27
JJXX
28
GG
SS
DD
VV
29
RRLL
MM
GG
30
SS
RR
TT
JJ
HH
PP
KK
GG
WW
XX
31
DD
RR
TT
VV
GG
PP
LL
AA
KK
NN
32
LLKK
SS
RR
TT
GG
AA
PP
VV
EE
33
VV
SS
34
TT
LL
GG
PP
SS
CC
WW
35
SSRR
MM
LL
AA
TT
GG
VV
HH
PP
36 37
SS
RR
GG
TT
PP
VV
LL
AA
EE
KK
38
HH
YY
DD
KK
NN
EE
39
SS
RR
II
XX
MM
NN
TT
KK
JJ
40 41
VV
RR
LL
AA
PP
HH
42
RR
KK
SS
AA
NN
TT
LL
JJ
FF
HH
YY
43
PP
RRJJ
44
RR
TT
SS
PP
LL
VV
JJ
GG
45
AA
GG
PP
SS
WW
LL
EE
CC
HH
FF
II
46
TT
GG
JJ
CC
YY
LL
RR
SS
NN
KK
MM
47
RR
GG
HH
CC
MM
SS
LL
NN
KK
YY
48
KK
GG
VV
RR
JJ
FF
WW
49
RR
LL
KKJJ
HH
CC
YY
50
LL
KK
JJ
CCVV
RR
PP
GG
FF
DD
AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
JJ
GG
DD
LL
52 53
RR
KK
LL
NN
PP
TT
GG
SS
HH
YY
54
RR
NN
JJ
VV
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
RRTT
KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
FF
EE
70
RR
VV
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
function logos
Genome
g
tRNA
genes
T
g
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g tRNA CIFs and Eukaryotic tRNA
Gene Clusters are Conserved tRNA Class Informative Features
(CIFs) are Grounded in Physics,
not Evolutionary Conservation
Outline
We often usefully visualize biological reactions
in isolation, outside of their cellular contexts …
X of the first pentapeptide side chain with theL-Ala at the fourth
position of another, or indirect which first requires the formation
of a connecting amino acid bridge. Indirect cross-linking is more
common in bacteria that haveL-Lys at position X and the structure
of the required intervening amino acid bridge can vary between
different genera and species[7,8]. The amino acids required for
bridge formation are typically derived from appropriately aminoa-
cylated-tRNA donor molecules. Utilization of these donor tRNA
molecules in peptidoglycan biosynthesis and how this relates to
antibiotic resistance, are discussed below.
2.1. MurM and MurN in Streptococcus pneumoniae
The peptidoglycan layer ofS. pneumoniaeis unusual in that it
contains a combination of both linear and branched muropeptides
within the structure. Branched muropeptides carry either anL-Ala-
L-Ala or anL-Ser-L-Ala dipeptide that is attached to the thirdL-Lys
of the pentapeptide side chain of Lipid II[9](Fig. 3). Formation of
this dipeptide substrate for indirect peptidoglycan cross-linking re-
quires the activities of the MurM and MurN tRNA-dependent ami-
noacyl-ligases. MurM and MurN are encoded within an operon by
thefibAandfibB(factors important inb-lactam resistance) genes,
respectively. In addition, MurMN activity is functionally conserved
across all strains of pneumococcus[10,11]. Selective inactivation of
these two genes has shown that the protein products act within a
specific order. MurM is responsible for the addition of eitherL-Ala
orL-Ser as the first amino acid of the cross-link and then MurN
invariably addsL-Ala as the second amino acid[12]. In both cases,
appropriately aminoacylated-tRNA species serve as the amino acid
donors for the reaction[13]. Provision of adequate substrates for
MurMN is currently thought to be achieved solely by the activities
of alanyl- and seryl-tRNA synthetases within this bacterium. No
MurMN-specific tRNA isoacceptors have been identified to date
that would explain how a balance between direction of these
aminoacylated-tRNA species into the protein and peptidoglycan
biosynthesis pathways is either established or maintained.
In 1990, prior to the identification of thefibABoperon, a link
was established between levels of indirect cross-linking in the
pneumococcal cell wall and penicillin resistance. At this time, it
was observed that a penicillin-resistant isolate from South Africa
had a highly branched peptidoglycan that could be co-transferred
with penicillin resistance to susceptible pneumococci[14,15].
Since then, many highly penicillin-resistant strains of pneumococ-
cus have also been shown to have an increased level of branched
muropeptides within their cell wall in comparison to penicillin-
sensitive isolates. Further investigation by insertion duplication
mutagenesis targeting thefibABoperon showed that inactivation
of MurMN had no significant effect on cell viability. Notably, inter-
ruption of thefibABoperon inS. pneumoniaestrain Pen6 caused a
marked reduction in penicillin resistance from 6 to 0.032lg ml
!1
.
This effect was also seen in other penicillin-resistant strains of
pneumococcus regardless of their genetic background, labeling
Fig. 1.Formation of an amino acid-tRNA pair by an aminoacyl-tRNA synthetase (aaRS). An amino acid (blue) is activated by an aaRS to form an aminoacyl-adenylate. This
process requires adenosine triphosphate (ATP) and results in the release of pyrophosphate (PPi). Following tRNA binding to the aaRS, the activated amino acid is transferred to
the 3
0
end of the tRNA molecule forming aminoacyl-tRNA with concurrent release of adenosine monophosphate (AMP). The aminoacyl-tRNA product is released from the
aaRS and is either subject to binding by elongation factor Tu (EF–Tu) for delivery to the ribosome or hijacking by other factors for diversion into biosynthetic pathways outside
of translation. Figure reproduced with permission from[2].
Fig. 2.Cellular biosynthetic pathways that utilize aminoacyl-tRNAs as substrates.
2896 J. Shepherd, M. Ibba / FEBS Letters 587 (2013) 2895–2904
in isolation, outside of their cellular contexts …
X of the first pentapeptide side chain with theL-Ala at the fourth
position of another, or indirect which first requires the formation
of a connecting amino acid bridge. Indirect cross-linking is more
common in bacteria that haveL-Lys at position X and the structure
of the required intervening amino acid bridge can vary between
different genera and species[7,8]. The amino acids required for
bridge formation are typically derived from appropriately aminoa-
cylated-tRNA donor molecules. Utilization of these donor tRNA
molecules in peptidoglycan biosynthesis and how this relates to
antibiotic resistance, are discussed below.
2.1. MurM and MurN in Streptococcus pneumoniae
The peptidoglycan layer ofS. pneumoniaeis unusual in that it
contains a combination of both linear and branched muropeptides
within the structure. Branched muropeptides carry either anL-Ala-
L-Ala or anL-Ser-L-Ala dipeptide that is attached to the thirdL-Lys
of the pentapeptide side chain of Lipid II[9](Fig. 3). Formation of
this dipeptide substrate for indirect peptidoglycan cross-linking re-
quires the activities of the MurM and MurN tRNA-dependent ami-
noacyl-ligases. MurM and MurN are encoded within an operon by
thefibAandfibB(factors important inb-lactam resistance) genes,
respectively. In addition, MurMN activity is functionally conserved
across all strains of pneumococcus[10,11]. Selective inactivation of
these two genes has shown that the protein products act within a
specific order. MurM is responsible for the addition of eitherL-Ala
orL-Ser as the first amino acid of the cross-link and then MurN
invariably addsL-Ala as the second amino acid[12]. In both cases,
appropriately aminoacylated-tRNA species serve as the amino acid
donors for the reaction[13]. Provision of adequate substrates for
MurMN is currently thought to be achieved solely by the activities
of alanyl- and seryl-tRNA synthetases within this bacterium. No
MurMN-specific tRNA isoacceptors have been identified to date
that would explain how a balance between direction of these
aminoacylated-tRNA species into the protein and peptidoglycan
biosynthesis pathways is either established or maintained.
In 1990, prior to the identification of thefibABoperon, a link
was established between levels of indirect cross-linking in the
pneumococcal cell wall and penicillin resistance. At this time, it
was observed that a penicillin-resistant isolate from South Africa
had a highly branched peptidoglycan that could be co-transferred
with penicillin resistance to susceptible pneumococci[14,15].
Since then, many highly penicillin-resistant strains of pneumococ-
cus have also been shown to have an increased level of branched
muropeptides within their cell wall in comparison to penicillin-
sensitive isolates. Further investigation by insertion duplication
mutagenesis targeting thefibABoperon showed that inactivation
of MurMN had no significant effect on cell viability. Notably, inter-
ruption of thefibABoperon inS. pneumoniaestrain Pen6 caused a
marked reduction in penicillin resistance from 6 to 0.032lg ml
!1
.
This effect was also seen in other penicillin-resistant strains of
pneumococcus regardless of their genetic background, labeling
Fig. 1.Formation of an amino acid-tRNA pair by an aminoacyl-tRNA synthetase (aaRS). An amino acid (blue) is activated by an aaRS to form an aminoacyl-adenylate. This
process requires adenosine triphosphate (ATP) and results in the release of pyrophosphate (PPi). Following tRNA binding to the aaRS, the activated amino acid is transferred to
the 3
0
end of the tRNA molecule forming aminoacyl-tRNA with concurrent release of adenosine monophosphate (AMP). The aminoacyl-tRNA product is released from the
aaRS and is either subject to binding by elongation factor Tu (EF–Tu) for delivery to the ribosome or hijacking by other factors for diversion into biosynthetic pathways outside
of translation. Figure reproduced with permission from[2].
Fig. 2.Cellular biosynthetic pathways that utilize aminoacyl-tRNAs as substrates.
2896 J. Shepherd, M. Ibba / FEBS Letters 587 (2013) 2895–2904
… but crowded cellular compartments are mixed by diffusion.
Every tRNA interacts with every aaRS…
Every tRNA interacts with every aaRS…
…while all tRNAs
must structurally
conform to the
ribosome, EF-Tu
etc.
Thus, tRNA genes evolve under conflicting constraints of
identity and conformity
Each tRNA class has
preferred protein
binding partners…
Movie credit: Ramakrishnan LabAARS structure figures: Hadd and Perona
must structurally
conform to the
ribosome, EF-Tu
etc.
Thus, tRNA genes evolve under conflicting constraints of
identity and conformity
Each tRNA class has
preferred protein
binding partners…
Movie credit: Ramakrishnan LabAARS structure figures: Hadd and Perona
Asn-tRNAs
Phe-tRNAs
His-tRNAs
Leu-tRNAs
Trp-tRNAs
Tyr-tRNAs
ATCG
N
N
N
N
N
N
F
F
F
F
F
F
H
H
H
H
H
H
L
L
L
L
L
L
W
W
W
W
W
Y
Y
Y
Y
Y
Y
The structural conformity of tRNAs
enables bioinformatic analysis of identity
(Gaps)
Variable Arm
Phe-tRNAs
His-tRNAs
Leu-tRNAs
Trp-tRNAs
Tyr-tRNAs
ATCG
N
N
N
N
N
N
F
F
F
F
F
F
H
H
H
H
H
H
L
L
L
L
L
L
W
W
W
W
W
Y
Y
Y
Y
Y
Y
The structural conformity of tRNAs
enables bioinformatic analysis of identity
(Gaps)
Variable Arm
6 T.D.Schneider
! SEQUENCE LOGO
--------- +++++++++
9876543210123456789
...................
1 GTATCACCGCCAGTGGTAT
2 ATACCACTGGCGGTGATAC
3 TCAACACCGCCAGAGATAA
4 TTATCTCTGGCGGTGTTGA
5 TTATCACCGCAGATGGTTA
6 TAACCATCTGCGGTGATAA
7 CTATCACCGCAAGGGATAA
8 TTATCCCTTGCGGTGATAG
9 CTAACACCGTGCGTGTTGA
10 TCAACACGCACGGTGTTAG
11 TTACCTCTGGCGGTGATAA
12 TTATCACCGCCAGAGGTAA
12 Lambda cI and cro binding sites
0
1
2
bits|
-9
G
A
CT
-8
A
CT
-7 A
-6
C
A
T
-5 C
-4
C
TA
-3
TC
-2
GT
C
-1
C
TG
0
TAG
C
1
G
AC
2
CA
G
3
AG
4
G
AT
5 G
6
T
G
A
7 T
8
T
GA
9
T
C
GA|
raw sequences
of binding sites
The height of each
stack of letters is the
information content
(sequence
conservation)
in bits per base
letter heights are
proportional to
frequency:
8/12 = 67% A
2/12 = 17% G
1/12 = 8% C
1/12 = 8% T
Minor Groove Faces Protein
Major Groove Faces Protein
Zero coordinate
for alignment
Error bar
for entire
stack of letters
Fig.1.2.Sequencelogoforthe6sequences(andtheircomplements)boundbyboth
thebacteriophage!cIrepressorandthecroproteins.Thesequencesaregiven5
!
to3
!
.ThemethodofcomputingthestackheightsisgiveninFig.1.1.
andminorgrooverespectively.UsingexperimentaldataIfoundthatthe
peaksofinformationcorrespondtoplaceswhereamajorgroovefacesthe
protein[20].(SeeFig.1.2foranexample.)
Sequence logos visualize information about
structure, conditioned by a common function
10 bp
10
bp
Schneider and Stephens (1990)
! SEQUENCE LOGO
--------- +++++++++
9876543210123456789
...................
1 GTATCACCGCCAGTGGTAT
2 ATACCACTGGCGGTGATAC
3 TCAACACCGCCAGAGATAA
4 TTATCTCTGGCGGTGTTGA
5 TTATCACCGCAGATGGTTA
6 TAACCATCTGCGGTGATAA
7 CTATCACCGCAAGGGATAA
8 TTATCCCTTGCGGTGATAG
9 CTAACACCGTGCGTGTTGA
10 TCAACACGCACGGTGTTAG
11 TTACCTCTGGCGGTGATAA
12 TTATCACCGCCAGAGGTAA
12 Lambda cI and cro binding sites
0
1
2
bits|
-9
G
A
CT
-8
A
CT
-7 A
-6
C
A
T
-5 C
-4
C
TA
-3
TC
-2
GT
C
-1
C
TG
0
TAG
C
1
G
AC
2
CA
G
3
AG
4
G
AT
5 G
6
T
G
A
7 T
8
T
GA
9
T
C
GA|
raw sequences
of binding sites
The height of each
stack of letters is the
information content
(sequence
conservation)
in bits per base
letter heights are
proportional to
frequency:
8/12 = 67% A
2/12 = 17% G
1/12 = 8% C
1/12 = 8% T
Minor Groove Faces Protein
Major Groove Faces Protein
Zero coordinate
for alignment
Error bar
for entire
stack of letters
Fig.1.2.Sequencelogoforthe6sequences(andtheircomplements)boundbyboth
thebacteriophage!cIrepressorandthecroproteins.Thesequencesaregiven5
!
to3
!
.ThemethodofcomputingthestackheightsisgiveninFig.1.1.
andminorgrooverespectively.UsingexperimentaldataIfoundthatthe
peaksofinformationcorrespondtoplaceswhereamajorgroovefacesthe
protein[20].(SeeFig.1.2foranexample.)
Sequence logos visualize information about
structure, conditioned by a common function
10 bp
10
bp
Schneider and Stephens (1990)
0
1
2
3
4
bits
-1H
1
WJ
K
I
C
E
R
L
V
T
D
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2
LRE
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3
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T
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4
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N
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12
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13
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1415
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16
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A
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17
17a
1819 20
T
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20a
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20bS
21
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J
ICL
22
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Q
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E
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N
23
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Q
Y
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24
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D
W
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A
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N
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25
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26
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27
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Q
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28
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29
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30
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31
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323334
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35
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A
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36
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L
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37
SV
R
Q
L
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P
38
ALP
39
M
C
QL
P
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R
A
I
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G
D
40
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IA
L
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41
HR
L
V
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42
F
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C
L
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D
E
W
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Y
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Q
43
M
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S
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T
A
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44
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QD
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T
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A
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45
C
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K
N
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46
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L
G
W
T
R
D
A
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V
F
J
I
H
K
P
N
M
47
LQ
48
A
C
V
G
49
E
A
K
T
V
R
C
Q
W
X
P
H
S
J
Y
F
G
L
I
D
M
N
50
K
J
Y
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GE
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W
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A
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L
M
I
51
V
N
C
J
L
A
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52 5354555657
X
IM
J
L
T
V
FY
D
C
W
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P
E
H
K
A
G
S
N
Q
58
H
59
AG
F
P
X
R
H
T
K
E
M
W
V
N
I
J
L
D
60
HW
6162
I
L
GK
R
63
R
G
ED
P
I
M
X
L
T
V
H
F
N
A
C
64
S
A
GI
Y
T
F
LR
Q
P
V
J
W
K
N
C
X
H
E
D
65
L
R
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C
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QK
66
VE
67
I
D
SM
G
V
F
T
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L
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H
W
Q
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A
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68
M
H
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C
T
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A
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69
TS
Q
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L
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K
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C
70
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QL
T
C
E
N
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M
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71
K
I
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D
R
L
T
G
H
N
72
L
R
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NP
73
FE
R
Q
S
N
D
W
0
1
2
3
4
bits
-1
1
L
V
R
NX
P
2
V
Q
J
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KR
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D
L
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F
N
G
T
3
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H
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N
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C
4
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L
N
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C
V
F
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5
G
Y
FS
V
M
C
L
P
K
H
N
T
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A
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6
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MW
D
JS
I
T
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G
N
Q
L
P
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C
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A
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7
G
L
RVY
89
GQE
10
SY
11
XQ
GL
Y
P
C
W
V
R
I
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M
K
F
A
T
J
H
N
D
12
LQ
S
Y
E
13
L
E
QG
P
H
X
I
T
D
A
W
V
K
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F
R
N
M
14Y
15
L
G
T
H
16
C
I
G
F
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D
Y
S
J
W
L
T
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M
Q
V
P
N
A
R
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17
S
VN
AL
M
T
RD
KP
X
17a
SPX
181920
V
X
P
E
H
K
L
T
Q
C
M
GS
Y
20a
L
R
P
I
V
X
YJ
M
WE
S
20b
LE
2122Y
23
RS
Q
GP
C
L
X
24
SX
Y
Q
G
25
D
YR
S
G
L
I
P
V
A
C
E
T
W
X
F
K
J
M
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N
26
PLQ
GJ
27
F
Y
X
N
C
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W
M
G
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L
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S
A
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28
I
J
M
V
N
H
L
TG
F
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S
A
R
P
Y
K
Q
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D
X
W
29
G
EKL
P
M
R
V
F
T
S
N
J
I
30
D
Q
MI
A
L
P
R
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G
J
E
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31
FQP
L
V
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A
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D
I
32
V
A
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M
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L
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3334
A
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35
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36
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3738
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39
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40
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A
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41
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T
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42
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P
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MG
Q
A
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43
I
T
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A
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GL
E
R
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M
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Y
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44
E
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45
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46
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47
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48
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49
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50
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51
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52
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535455
T
56575859
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60
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616263
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64
V
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A
F
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65
E
CG
Q
V
A
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66
Y
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67
G
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68
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69
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70
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71
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73
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1
2
3
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bits
-1
1
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2
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3
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N
MVH
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4
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L
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9
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10
A
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11
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12
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14
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A
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W
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16
L
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17
LF
CH
RM
17a
TX
18 19 20
LKR
20a
G
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20b
LY
21
C
LY
N
V
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22
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26
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27
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28
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29
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A
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30
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31
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V
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S
P
A
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T
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M
K
F
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32
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M
33 34
L
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35
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36
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S
C
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37
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38
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39
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40
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41
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42
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43
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47
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48
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49
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52
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53
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R
54 55 56 57
E
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58 59
D
L
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M
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60
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L
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61 62
G
JR
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63
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64
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65
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66
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67
K
M
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V
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L
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68
V
A
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N
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69
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70
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71
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72
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X
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73
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V 0
1
2
3
4
bits
-1
1
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2
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M
Q
X
G
D
A
H
5
E
R
A
JC
H
T
S
N
L
F
D
K
M
P
Y
V
Q
G
X
W
6
G
HA
JR
T
K
Q
E
L
V
C
I
M
Y
N
S
F
X
7
Y
W
V
A
K
T
Q
F
H
C
G
X
R
L
P
N
S
J
M
D
I
8
CY
9
T
I
ER
Q
G
PY
X
L
C
S
10
Y
S
G
L
P
R
A
C
Q
W
X
T
V
H
I
F
E
J
M
N
K
D
11
SX
Y
Q
12
RS
Q
GP
C
L
X
13
L
Y
C
S
14 15
L
JG
E
Q
Y
WR
C
H
T
V
X
A
S
I
F
D
K
P
N
M
16
R
P
V
H
LK
T
M
Q
F
G
I
17
17a
18 19 20
L
IFV
A
20a
M
20bS
21
V
NY
CL
22
L
Y
CQ
G
N
X
P
E
R
V
W
A
T
H
F
D
I
M
K
J
23
LGQ
S
Y
E
24
XQ
G
L
Y
P
W
V
I
S
R
T
A
F
D
K
J
M
C
H
N
25
SY
26
F
K
N
C
Q
J
TM
P
G
R
V
W
S
Y
L
D
A
I
E
H
X
27
VI
XA
P
LE
T
R
S
W
G
N
K
Y
M
Q
F
J
C
28
S
P
R
E
Q
G
K
I
N
M
T
L
A
Y
F
J
29
X
G
VA
KT
P
J
R
E
H
S
L
F
Y
Q
M
C
D
W
30
V
T
E
J
G
S
R
P
L
A
I
M
Q
D
Y
X
W
C
NK
F
H
31
G
R
Q
H
K
MA
L
E
P
N
V
J
X
32 33 34
L
A
P
T
V
G
S
Y
C
H
I
N
D
F
35
S
A
P
T
36
R
L
H
Q
P
37
GXI
R
FQ
L
P
H
38
LG
AP
39
FQP
L
V
T
S
R
E
A
G
D
I
40
D
QMI
A
L
P
R
S
G
J
E
T
V
41
EKL
P
M
R
V
F
T
S
N
J
I
42
I
J
M
V
N
H
L
TG
F
C
S
A
R
P
Y
K
Q
E
X
D
W
43
N
F
J
Y
X
Q
KW
M
G
R
E
P
L
T
S
V
A
H
I
D
44
LNH
E
Q
A
I
D
R
T
W
G
V
K
M
P
C
F
45
C
F
Q
S
E
L
Y
M
G
V
N
R
A
P
K
W
I
X
T
H
D
J
46
S
E
Y
Q
G
L
H
R
T
D
V
X
W
A
K
J
I
M
F
P
N
47
R
ELQ
48
CY
E
49
E
CQ
V
AK
R
H
Y
X
T
S
F
G
J
W
M
L
P
I
D
N
50
K
N
XC
G
Q
S
P
W
H
R
V
T
J
L
A
F
E
M
I
51
I
N
H
F
XC
A
R
P
V
L
K
J
T
W
M
E
G
D
S
52 53 54E
55 56 57
X
I
M
H
V
T
YJ
P
C
L
F
W
S
R
G
D
K
E
A
Q
N
58K
59
A
GC
Q
E
P
R
N
Y
V
K
T
M
H
W
L
J
I
D
6061 62
L
TG
I
R
H
63
JX
Y
P
G
I
E
K
V
M
T
L
H
N
A
C
F
64
M
SE
F
K
T
L
G
Y
H
J
W
V
R
P
N
Q
X
C
D
65
L
X
P
W
G
R
M
V
F
J
T
S
Y
H
K
C
Q
66
G
L
RVY
67
X
MD
S
I
V
N
T
Q
L
G
K
P
C
H
W
A
E
R
J
68
G
F
M
SV
C
L
P
K
H
N
T
A
J
R
E
I
69
Q
G
MST
Y
L
N
I
K
C
V
F
J
R
E
70
S
P
QJE
R
L
H
T
M
F
N
C
X
71
V
Q
J
W
KR
E
D
L
H
F
N
G
T
72
V
R
N
FP
73
X
T
FE
R
Q
N
S
D
W 0
1
2
3
4
bits
-1
1
L
IXN
Q
2
LK
H
E
R
3
V
L
XM
SR
E
T
4
X
G
L
P
J
T
SR
F
M
N
V
K
I
5
EV
P
QA
M
J
RT
N
L
I
D
C
H
6
Y
MR
S
CK
V
Q
A
L
H
I
P
T
G
D
J
W
7
G
PF
Q
M
S
LR
WT
C
A
H
K
V
E
8
Y
C
X
Q
R
N
P
VW
S
T
M
F
G
E
A
D
K
L
J
H
I
9G
Y
10
PY
S
11
I
R
V
W
C
P
YL
G
QE
12
L
Y
S
PQ
G
T
R
V
A
W
I
J
F
H
M
N
K
D
13
T
X
I
H
PG
QE
14 15
T
VE
16
E
XR
A
N
P
V
Q
I
M
G
T
Y
K
F
L
W
S
J
C
D
H
17
Q
G
PN
V
J
T
M
K
F
R
L
D
H
W
A
I
17a
G
IF
W
XP
18 19 20
AV
Y
S
GM
C
F
Q
T
K
L
H
E
P
I
X
D
W
J
N
20a
L
X
T
N
K
CG
W
V
PY
S
E
M
J
H
I
R
D
20b
RE
S
21
LC
22
P
X
NSG
Y
23
LR
Q
VY
G
C
T
S
24X
25
A
V
P
I
S
L
G
RD
26
L
M
H
S
J
Q
27
SYC
M
D
A
J
T
L
I
Q
H
KN
R
V
E
W
P
F
GX
28
X
QD
J
E
K
M
G
S
A
F
L
T
P
R
H
N
V
29
I
S
M
L
XG
QY
R
H
N
V
P
T
K
A
30
L
G
V
JP
31
AM
GR
L
T
CH
P
S
Q
W
32
Y
X
WM
SR
C
I
E
Q
P
F
L
A
G
H
T
V
33 34
S
R
G
T
P
V
L
A
K
Q
E
35
D
Y
K
Q
H
E
N
36
S
R
J
X
I
K
M
N
T
37 38
G
HR
P
E
A
V
Q
L
39
G
V
WJ
E
S
P
A
Q
R
L
T
N
H
C
M
K
F
Y
40
G
P
E
L
T
R
QS
41
T
F
A
Y
K
Q
P
H
V
R
L
E
G
X
42
Q
VNP
RE
K
A
L
F
T
S
H
G
C
J
M
I
43
V
P
F
E
G
T
R
S
L
W
K
Q
M
Y
J
N
C
44
PWM
E
VG
K
C
R
Q
S
Y
L
N
J
45
T
XY
K
L
P
A
RN
Q
V
G
C
M
F
46
LS
Q
Y
47
L
S
Y
Q
G
X
R
P
W
T
H
D
V
A
I
K
J
F
N
M
48
A
HC
G
W
R
Q
J
L
49
LR
H
W
C
K
50
E
C
V
I
T
R
L
G
W
P
H
X
N
A
S
K
Q
Y
51
R
K
T
L
J
P
V
I
XN
H
Q
Y
F
52
G
JR
P
HT
53 54 55 56
A
57 58 59
P
J
WH
M
Q
R
L
K
Y
N
T
V
G
F
E
A
C
60
D
A
H
V
W
KT
N
F
L
Q
G
M
R
S
C
Y
X
E
P
I
J
61
GH
R
62
XF
S
E
K
J
H
W
R
M
G
C
A
P
L
I
V
T
63
S
G
AT
Y
J
L
Q
K
V
N
W
P
R
I
X
64
C
QI
N
Y
F
P
L
R
A
K
W
G
V
J
S
T
M
65
Q
Y
IW
H
KF
T
X
S
P
C
L
R
V
A
G
E
D
66
CHV
JR
N
M
P
L
K
X
G
A
S
I
F
E
T
Q
Y
W
67
R
AN
C
G
W
P
X
F
L
S
D
T
Q
K
V
M
Y
68
R
G
T
P
V
N
A
D
Q
H
Y
M
L
C
F
S
69
V
L
G
Q
X
T
CM
K
S
H
W
D
P
70
LIE
N
FMVH
R
Y
S
A
71
S
P
X
M
DR
K
72
R
XSK
V
I
LJ
E
M
H
W
73
L
I
S
T
QG
C 0
1
2
3
4
bits
-1
1
L
V
R
NX
P
2
V
Q
J
W
KR
E
D
L
H
F
N
G
T
3
S
Q
PJR
E
L
H
T
M
F
N
X
C
4
G
Q
MST
Y
L
N
I
K
C
V
F
J
R
E
5
G
Y
FS
V
M
C
L
P
K
H
N
T
I
A
R
J
E
6
X
MW
D
JS
I
T
V
G
N
Q
L
P
K
C
H
A
E
R
7
G
L
RVY
89
GQE
10
SY
11
XQ
GL
Y
P
C
W
V
R
I
S
M
K
F
A
T
J
H
N
D
12
LQ
S
Y
E
13
L
E
QG
P
H
X
I
T
D
A
W
V
K
J
F
R
N
M
14Y
15
L
G
T
H
16
C
I
G
F
H
D
Y
S
J
W
L
T
K
M
Q
V
P
N
A
R
X
E
17
S
VN
AL
M
T
RD
KP
X
17a
SPX
18 19 20
V
X
P
E
H
K
L
T
Q
C
M
GS
Y
20a
L
R
P
I
V
X
YJ
M
WE
S
20b
LE
21 22Y
23
RS
Q
GP
C
L
X
24
SX
Y
Q
G
25
D
YR
S
G
L
I
P
V
A
C
E
T
W
X
F
K
J
M
Q
H
N
26
PLQ
GJ
27
F
Y
X
N
C
Q
K
W
M
G
R
E
P
T
L
V
S
A
H
I
D
28
I
J
M
V
N
H
L
TG
F
C
S
A
R
P
Y
K
Q
E
D
X
W
29
G
EKL
P
M
R
V
F
T
S
N
J
I
30
D
Q
MI
A
L
P
R
S
G
J
E
T
V
31
FQP
L
V
T
S
R
E
A
G
D
I
32
V
A
TH
M
G
L
P
F
Q
E
I
C
R
S
W
X
Y
K
J
N
D
33 34
A
S
G
V
E
P
T
Q
L
K
R
X
J
M
W
35
SC
W
G
R
36
D
A
G
E
V
37 38
I
G
XL
R
A
S
Y
H
Q
E
V
D
39
G
R
Q
H
K
AM
L
E
P
N
V
J
X
40
V
T
E
J
S
G
R
P
L
A
I
M
Q
D
F
W
X
Y
K
H
N
C
41
X
G
VAP
T
J
R
E
H
S
L
F
Y
M
Q
C
D
W
42
SI
P
R
K
MG
Q
A
T
N
L
Y
F
J
43
I
T
V
XCS
A
P
GL
E
R
W
N
M
J
Y
K
Q
F
44
E
K
H
J
C
W
R
N
GL
Y
S
Q
45
P
Q
YGC
46
Q
Y
L
S
47
G
K
H
I
A
S
V
D
T
W
P
Q
R
Y
X
48
L
JE
Q
Y
R
G
W
C
H
T
V
X
A
S
I
F
D
K
P
N
M
49
L
X
P
W
G
R
M
V
F
J
T
S
Y
H
K
C
Q
50
M
SE
F
K
T
L
G
Q
P
Y
H
J
W
R
V
N
X
C
D
51
J
X
YP
G
I
K
E
F
V
M
N
T
L
H
A
C
52
L
M
TG
I
R
H
53 54 55
T
56 57 58 59
K
V
X
N
I
G
R
C
T
F
A
LH
E
60
SR
M
G
Q
L
FN
T
K
H
W
V
A
D
61 62 63
I
N
F
XC
H
R
A
P
V
L
W
K
J
T
M
E
G
S
D
64
V
K
XS
C
W
G
Q
T
R
M
P
H
L
A
F
E
I
65
E
CG
Q
V
A
K
R
H
L
S
P
Y
T
X
F
J
W
M
I
N
66
Y
T
W
V
AK
Q
F
I
H
S
C
G
X
R
L
M
P
N
J
D
67
G
A
J
R
T
K
Q
E
L
V
C
I
M
Y
N
S
X
F
68
R
A
J
H
C
TS
N
L
F
D
M
K
P
Y
V
Q
G
X
W
69
R
V
FJC
P
N
D
L
Y
W
T
H
S
M
Q
X
G
A
70
M
SRH
Y
E
L
J
V
Q
P
I
G
D
K
W
71
T
G
N
L
F
D
RE
K
M
W
J
Q
I
X
V
P
S
Y
C
A
72
P
HM
EJ
L
I
V
K
S
F
R
C
A
Y
D
T
G
73
A
P
N
HX
U
C
G
A
Rhizobiales + Rhodobacteriaceae + Caulobacterales +
Hyphomonadaceae (RRCH)
Rickettsiales +Sphingomonadales + Rhodospirialles (RSR)
U
C
G
A
genome
functionally classified tRNAs
X R C E
J E Y
Score each tRNA
sequence for taxon-specific
functional Class-Informative-
Features (CIFs)
Average scores across tRNAs.
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM 0
1
2
3
4
bits
-1
1
C
I
K
JW
2E
K
3
VKR
S
Y
4
I
M
L
R
D
Y
J
S
T
H
P
W
5
K
N
G
D
A
C
V
P
T
H
Y
M
L
Q
F
S
6
K
C
R
S
X
E
F
L
N
T
G
Y
I
P
M
V
7
I
D
H
W
J
P
Y
V
R
X
E
Q
F
G
L
N
T
C
K
S
A
89
X
C
Q
P
G
T
V
R
A
W
I
J
F
H
M
N
K
D
10
SG
Y
L
11X
12
PT
G
SC
13
L
C
Q
1415
K
VH
T
R
Q
J
C
G
W
L
16
T
H
C
V
R
I
L
M
Y
Q
A
S
E
17
L
K
H
M
R
17a
SR
P
1819R
20
T
AR
20aL
20b
RY
21
L
CI
J
H
T
E
S
R
Q
X
Y
W
V
G
K
D
F
N
P
A
M
22
T
GY
Q
S
L
C
23
YQ
S
P
G
T
V
A
W
R
H
I
D
F
M
N
J
K
24
W
D
T
G
Q
LE
25S
26
X
D
L
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V
A
K
R
H
L
S
P
Y
T
X
F
J
W
M
I
N
66
Y
T
W
V
AK
Q
F
I
H
S
C
G
X
R
L
M
P
N
J
D
67
G
A
J
R
T
K
Q
E
L
V
C
I
M
Y
N
S
X
F
68
R
A
J
H
C
TS
N
L
F
D
M
K
P
Y
V
Q
G
X
W
69
R
V
FJC
P
N
D
L
Y
W
T
H
S
M
Q
X
G
A
70
M
SRH
Y
E
L
J
V
Q
P
I
G
D
K
W
71
T
G
N
L
F
D
RE
K
M
W
J
Q
I
X
V
P
S
Y
C
A
72
P
HM
EJ
L
I
V
K
S
F
R
C
A
Y
D
T
G
73
A
P
N
HX0
1
2
3
4
bits
-1
1
L
IXN
Q
2
LK
H
E
R
3
V
L
XM
SR
E
T
4
X
G
L
P
J
T
SR
F
M
N
V
K
I
5
EV
P
QA
M
J
RT
N
L
I
D
C
H
6
Y
MR
S
CK
V
Q
A
L
H
I
P
T
G
D
J
W
7
G
PF
Q
M
S
LR
WT
C
A
H
K
V
E
8
Y
C
X
Q
R
N
P
VW
S
T
M
F
G
E
A
D
K
L
J
H
I
9G
Y
10
PY
S
11
I
R
V
W
C
P
YL
G
QE
12
L
Y
S
PQ
G
T
R
V
A
W
I
J
F
H
M
N
K
D
13
T
X
I
H
PG
QE
14 15
T
VE
16
E
XR
A
N
P
V
Q
I
M
G
T
Y
K
F
L
W
S
J
C
D
H
17
Q
G
PN
V
J
T
M
K
F
R
L
D
H
W
A
I
17a
G
IF
W
XP
18 19 20
AV
Y
S
GM
C
F
Q
T
K
L
H
E
P
I
X
D
W
J
N
20a
L
X
T
N
K
CG
W
V
PY
S
E
M
J
H
I
R
D
20b
RE
S
21
LC
22
P
X
NSG
Y
23
LR
Q
VY
G
C
T
S
24X
25
A
V
P
I
S
L
G
RD
26
L
M
H
S
J
Q
27
SYC
M
D
A
J
T
L
I
Q
H
KN
R
V
E
W
P
F
GX
28
X
QD
J
E
K
M
G
S
A
F
L
T
P
R
H
N
V
29
I
S
M
L
XG
QY
R
H
N
V
P
T
K
A
30
L
G
V
JP
31
AM
GR
L
T
CH
P
S
Q
W
32
Y
X
WM
SR
C
I
E
Q
P
F
L
A
G
H
T
V
33 34
S
R
G
T
P
V
L
A
K
Q
E
35
D
Y
K
Q
H
E
N
36
S
R
J
X
I
K
M
N
T
37 38
G
HR
P
E
A
V
Q
L
39
G
V
WJ
E
S
P
A
Q
R
L
T
N
H
C
M
K
F
Y
40
G
P
E
L
T
R
QS
41
T
F
A
Y
K
Q
P
H
V
R
L
E
G
X
42
Q
VNP
RE
K
A
L
F
T
S
H
G
C
J
M
I
43
V
P
F
E
G
T
R
S
L
W
K
Q
M
Y
J
N
C
44
PWM
E
VG
K
C
R
Q
S
Y
L
N
J
45
T
XY
K
L
P
A
RN
Q
V
G
C
M
F
46
LS
Q
Y
47
L
S
Y
Q
G
X
R
P
W
T
H
D
V
A
I
K
J
F
N
M
48
A
HC
G
W
R
Q
J
L
49
LR
H
W
C
K
50
E
C
V
I
T
R
L
G
W
P
H
X
N
A
S
K
Q
Y
51
R
K
T
L
J
P
V
I
XN
H
Q
Y
F
52
G
JR
P
HT
53 54 55 56
A
57 58 59
P
J
WH
M
Q
R
L
K
Y
N
T
V
G
F
E
A
C
60
D
A
H
V
W
KT
N
F
L
Q
G
M
R
S
C
Y
X
E
P
I
J
61
GH
R
62
XF
S
E
K
J
H
W
R
M
G
C
A
P
L
I
V
T
63
S
G
AT
Y
J
L
Q
K
V
N
W
P
R
I
X
64
C
QI
N
Y
F
P
L
R
A
K
W
G
V
J
S
T
M
65
Q
Y
IW
H
KF
T
X
S
P
C
L
R
V
A
G
E
D
66
CHV
JR
N
M
P
L
K
X
G
A
S
I
F
E
T
Q
Y
W
67
R
AN
C
G
W
P
X
F
L
S
D
T
Q
K
V
M
Y
68
R
G
T
P
V
N
A
D
Q
H
Y
M
L
C
F
S
69
V
L
G
Q
X
T
CM
K
S
H
W
D
P
70
LIE
N
FMVH
R
Y
S
A
71
S
P
X
M
DR
K
72
R
XSK
V
I
LJ
E
M
H
W
73
L
I
S
T
QG
C 0
1
2
3
4
bits
-1
1
L
V
R
NX
P
2
V
Q
J
W
KR
E
D
L
H
F
N
G
T
3
S
Q
PJR
E
L
H
T
M
F
N
X
C
4
G
Q
MST
Y
L
N
I
K
C
V
F
J
R
E
5
G
Y
FS
V
M
C
L
P
K
H
N
T
I
A
R
J
E
6
X
MW
D
JS
I
T
V
G
N
Q
L
P
K
C
H
A
E
R
7
G
L
RVY
89
GQE
10
SY
11
XQ
GL
Y
P
C
W
V
R
I
S
M
K
F
A
T
J
H
N
D
12
LQ
S
Y
E
13
L
E
QG
P
H
X
I
T
D
A
W
V
K
J
F
R
N
M
14Y
15
L
G
T
H
16
C
I
G
F
H
D
Y
S
J
W
L
T
K
M
Q
V
P
N
A
R
X
E
17
S
VN
AL
M
T
RD
KP
X
17a
SPX
18 19 20
V
X
P
E
H
K
L
T
Q
C
M
GS
Y
20a
L
R
P
I
V
X
YJ
M
WE
S
20b
LE
21 22Y
23
RS
Q
GP
C
L
X
24
SX
Y
Q
G
25
D
YR
S
G
L
I
P
V
A
C
E
T
W
X
F
K
J
M
Q
H
N
26
PLQ
GJ
27
F
Y
X
N
C
Q
K
W
M
G
R
E
P
T
L
V
S
A
H
I
D
28
I
J
M
V
N
H
L
TG
F
C
S
A
R
P
Y
K
Q
E
D
X
W
29
G
EKL
P
M
R
V
F
T
S
N
J
I
30
D
Q
MI
A
L
P
R
S
G
J
E
T
V
31
FQP
L
V
T
S
R
E
A
G
D
I
32
V
A
TH
M
G
L
P
F
Q
E
I
C
R
S
W
X
Y
K
J
N
D
33 34
A
S
G
V
E
P
T
Q
L
K
R
X
J
M
W
35
SC
W
G
R
36
D
A
G
E
V
37 38
I
G
XL
R
A
S
Y
H
Q
E
V
D
39
G
R
Q
H
K
AM
L
E
P
N
V
J
X
40
V
T
E
J
S
G
R
P
L
A
I
M
Q
D
F
W
X
Y
K
H
N
C
41
X
G
VAP
T
J
R
E
H
S
L
F
Y
M
Q
C
D
W
42
SI
P
R
K
MG
Q
A
T
N
L
Y
F
J
43
I
T
V
XCS
A
P
GL
E
R
W
N
M
J
Y
K
Q
F
44
E
K
H
J
C
W
R
N
GL
Y
S
Q
45
P
Q
YGC
46
Q
Y
L
S
47
G
K
H
I
A
S
V
D
T
W
P
Q
R
Y
X
48
L
JE
Q
Y
R
G
W
C
H
T
V
X
A
S
I
F
D
K
P
N
M
49
L
X
P
W
G
R
M
V
F
J
T
S
Y
H
K
C
Q
50
M
SE
F
K
T
L
G
Q
P
Y
H
J
W
R
V
N
X
C
D
51
J
X
YP
G
I
K
E
F
V
M
N
T
L
H
A
C
52
L
M
TG
I
R
H
53 54 55
T
56 57 58 59
K
V
X
N
I
G
R
C
T
F
A
LH
E
60
SR
M
G
Q
L
FN
T
K
H
W
V
A
D
61 62 63
I
N
F
XC
H
R
A
P
V
L
W
K
J
T
M
E
G
S
D
64
V
K
XS
C
W
G
Q
T
R
M
P
H
L
A
F
E
I
65
E
CG
Q
V
A
K
R
H
L
S
P
Y
T
X
F
J
W
M
I
N
66
Y
T
W
V
AK
Q
F
I
H
S
C
G
X
R
L
M
P
N
J
D
67
G
A
J
R
T
K
Q
E
L
V
C
I
M
Y
N
S
X
F
68
R
A
J
H
C
TS
N
L
F
D
M
K
P
Y
V
Q
G
X
W
69
R
V
FJC
P
N
D
L
Y
W
T
H
S
M
Q
X
G
A
70
M
SRH
Y
E
L
J
V
Q
P
I
G
D
K
W
71
T
G
N
L
F
D
RE
K
M
W
J
Q
I
X
V
P
S
Y
C
A
72
P
HM
EJ
L
I
V
K
S
F
R
C
A
Y
D
T
G
73
A
P
N
HX
U
C
G
A
Rhizobiales + Rhodobacteriaceae + Caulobacterales +
Hyphomonadaceae (RRCH)
Rickettsiales +Sphingomonadales + Rhodospirialles (RSR)
U
C
G
A
genome
functionally classified tRNAs
X R C E
J E Y
Score each tRNA
sequence for taxon-specific
functional Class-Informative-
Features (CIFs)
Average scores across tRNAs.
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM 0
1
2
3
4
bits
-1
1
C
I
K
JW
2E
K
3
VKR
S
Y
4
I
M
L
R
D
Y
J
S
T
H
P
W
5
K
N
G
D
A
C
V
P
T
H
Y
M
L
Q
F
S
6
K
C
R
S
X
E
F
L
N
T
G
Y
I
P
M
V
7
I
D
H
W
J
P
Y
V
R
X
E
Q
F
G
L
N
T
C
K
S
A
89
X
C
Q
P
G
T
V
R
A
W
I
J
F
H
M
N
K
D
10
SG
Y
L
11X
12
PT
G
SC
13
L
C
Q
1415
K
VH
T
R
Q
J
C
G
W
L
16
T
H
C
V
R
I
L
M
Y
Q
A
S
E
17
L
K
H
M
R
17a
SR
P
1819R
20
T
AR
20aL
20b
RY
21
L
CI
J
H
T
E
S
R
Q
X
Y
W
V
G
K
D
F
N
P
A
M
22
T
GY
Q
S
L
C
23
YQ
S
P
G
T
V
A
W
R
H
I
D
F
M
N
J
K
24
W
D
T
G
Q
LE
25S
26
X
D
L
A
S
HG
W
JI
C
E
R
M
P
Q
T
V
Y
F
N
K
27
A
G
C
J
X
Y
S
L
R
T
M
K
F
Q
N
28
A
M
V
J
R
T
D
S
G
N
L
C
F
I
29
K
TA
D
S
W
V
P
M
R
L
E
H
Y
X
G
30
S
T
31
VH
SP
N
Q
A
R
L
T
M
K
C
Y
F
32
AM
3334R
35
X
V
I
F
J
M
L
36
L
S
C
Y
F
W
37
LQ
R
V
S
E
Y
A
X
T
W
DI
F
G
J
M
N
K
C
38
D
HL
AP
T
G
S
R
Y
W
X
F
K
J
M
C
I
N
39
VI
A
R
T
LP
H
Q
S
W
40
T
41
L
S
HF
R
J
D
N
P
T
V
A
K
42
H
I
D
S
M
W
J
G
T
A
L
N
R
K
P
V
43
LQJ
N
S
T
E
G
K
F
P
D
W
A
V
R
H
X
44
L
SM
C
N
K
P
J
G
E
V
H
W
R
A
I
T
D
X
45
R
A
W
P
T
M
N
L
J
V
S
G
C
Q
E
46
S
T
W
Y
Q
L
G
C
E
47
L
Q
DIY
48
G
A
V
R
TD
49
IL
G
F
P
Y
J
S
K
H
X
T
W
C
R
V
A
E
50
RL
I
W
V
X
T
Q
K
A
Y
S
G
51
AT
W
N
K
V
L
P
R
X
Y
J
Q
I
52
S
QG
A
W
K
Y
RD
P
V
H
L
T
I
5354555657
SG
AK
H
E
P
R
W
C
D
Y
F
V
T
L
J
M
I
X
5859
CL
F
W
A
I
J
V
E
H
T
G
N
K
M
R
Q
X
P
Y
S
60
JHW
6162
M
R
N
T
K
63
T
LP
I
K
R
Q
V
N
X
Y
F
64
VM
Q
L
I
P
D
H
X
A
R
W
T
C
K
G
N
Y
F
S
J
65
Q
T
66
S
LK
A
R
T
V
E
67
M
F
K
V
E
R
A
P
Q
L
W
H
T
J
I
G
D
68
S
A
NJ
T
H
R
K
L
F
I
69
TN
C
R
K
S
V
F
E
I
M
70
S
RE
C
T
K
71
TR
E
72
X
N
Q
73
S
N
QR
E
T
F
Y
X
V
J
I
A
K
P
M
L
0
1
2
3
4
bits
-1
1
X
Q
N
2
TL
R
E
3
S
G
RE
C
T
K
4
L
TK
R
S
E
F
V
M
I
N
C
5
SJ
A
T
N
H
R
K
L
F
I
6
V
F
E
M
R
K
P
Q
A
L
T
H
I
G
J
W
D
7
S
LK
A
R
T
V
E
89
10S
11
S
V
W
D
M
T
Y
G
Q
LE
12
YQ
S
P
G
T
V
A
W
R
H
I
D
F
M
N
J
K
13
L
SV
D
R
T
N
G
QE
1415
A
R
D
C
V
16
P
I
E
X
M
R
A
Q
V
S
T
N
G
L
W
J
K
Y
H
F
D
C
17
PG
N
X
T
K
F
J
L
V
M
R
A
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ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM 0
1
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ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
0
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M
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2
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K
D
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3
S
T
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NR
K
L
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4
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CR
N
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L
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5
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N
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L
A
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V
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6
RH
J
K
T
L
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M
F
Y
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7
V
T
A
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RC
LN
G
F
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X
Y
P
J
W
H
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89
RV
Q
T
GP
C
X
S
Y
L
1011
QS
12
V
G
SCP
L
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13
TC
L
S
Y
1415
W
G
C
J
Q
R
T
H
V
A
D
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E
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X
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I
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M
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16
R
K
F
PV
J
A
M
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G
I
17
17a
1819 20
T
KVA
20a
C
20bS
21
H
J
ICL
22
S
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G
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M
E
D
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V
A
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23
L
T
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Y
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24
L
Q
G
ST
D
W
R
Y
V
A
P
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F
H
M
N
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C
25
S
Y
26
N
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Y
J
C
V
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T
G
M
R
E
W
I
H
A
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L
D
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27
PR
X
A
F
G
T
L
N
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K
Q
J
M
Y
C
28
P
H
I
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L
A
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E
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F
M
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29
KA
Y
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M
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W
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C
30
J
E
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LA
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M
H
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31
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K
A
M
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L
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323334
L
P
A
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35
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A
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36
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L
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37
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38
ALP
39
M
C
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P
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40
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L
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41
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L
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42
F
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C
L
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A
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D
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43
M
NY
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44
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L
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A
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45
C
S
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46
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L
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D
A
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F
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H
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47
LQ
48
A
C
V
G
49
E
A
K
T
V
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C
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P
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Y
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N
50
K
J
Y
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GE
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A
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L
M
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51
V
N
C
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L
A
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D
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52 5354555657
X
IM
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L
T
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FY
D
C
W
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A
G
S
N
Q
58
H
59
AG
F
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M
W
V
N
I
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L
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60
HW
6162
I
L
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63
R
G
ED
P
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L
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N
A
C
64
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A
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Y
T
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LR
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P
V
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W
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N
C
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65
L
R
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66
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67
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D
SM
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V
F
T
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L
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68
M
H
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69
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70
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71
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G
H
N
72
L
R
E
NP
73
FE
R
Q
S
N
D
W
0
1
2
3
4
bits
-1
1
L
V
R
NX
P
2
V
Q
J
W
KR
E
D
L
H
F
N
G
T
3
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4
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5
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7
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89
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10
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11
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Y
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12
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13
L
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15
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16
C
I
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W
L
T
K
M
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V
P
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A
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17
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VN
AL
M
T
RD
KP
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17a
SPX
181920
V
X
P
E
H
K
L
T
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C
M
GS
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20a
L
R
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I
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WE
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20b
LE
2122Y
23
RS
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GP
C
L
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24
SX
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25
D
YR
S
G
L
I
P
V
A
C
E
T
W
X
F
K
J
M
Q
H
N
26
PLQ
GJ
27
F
Y
X
N
C
Q
K
W
M
G
R
E
P
T
L
V
S
A
H
I
D
28
I
J
M
V
N
H
L
TG
F
C
S
A
R
P
Y
K
Q
E
D
X
W
29
G
EKL
P
M
R
V
F
T
S
N
J
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30
D
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MI
A
L
P
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31
FQP
L
V
T
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R
E
A
G
D
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32
V
A
TH
M
G
L
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3334
A
S
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35
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W
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36
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A
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3738
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39
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40
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T
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A
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41
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T
J
R
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M
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C
D
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42
SI
P
R
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MG
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A
T
N
L
Y
F
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43
I
T
V
XCS
A
P
GL
E
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W
N
M
J
Y
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44
E
K
H
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C
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N
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45
P
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46
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Y
L
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47
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W
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48
L
JE
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Y
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G
W
C
H
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A
S
I
F
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P
N
M
49
L
X
P
W
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T
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H
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50
M
SE
F
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L
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W
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C
D
51
J
X
YP
G
I
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E
F
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M
N
T
L
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52
L
M
TG
I
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535455
T
56575859
K
V
X
N
I
G
R
C
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F
A
LH
E
60
SR
M
G
Q
L
FN
T
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H
W
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616263
I
N
F
XC
H
R
A
P
V
L
W
K
J
T
M
E
G
S
D
64
V
K
XS
C
W
G
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T
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M
P
H
L
A
F
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65
E
CG
Q
V
A
K
R
H
L
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P
Y
T
X
F
J
W
M
I
N
66
Y
T
W
V
AK
Q
F
I
H
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C
G
X
R
L
M
P
N
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D
67
G
A
J
R
T
K
Q
E
L
V
C
I
M
Y
N
S
X
F
68
R
A
J
H
C
TS
N
L
F
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M
K
P
Y
V
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X
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69
R
V
FJC
P
N
D
L
Y
W
T
H
S
M
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X
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A
70
M
SRH
Y
E
L
J
V
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P
I
G
D
K
W
71
T
G
N
L
F
D
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K
M
W
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I
X
V
P
S
Y
C
A
72
P
HM
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L
I
V
K
S
F
R
C
A
Y
D
T
G
73
A
P
N
HX0
1
2
3
4
bits
-1
1
L
IXN
Q
2
LK
H
E
R
3
V
L
XM
SR
E
T
4
X
G
L
P
J
T
SR
F
M
N
V
K
I
5
EV
P
QA
M
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RT
N
L
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6
Y
MR
S
CK
V
Q
A
L
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T
G
D
J
W
7
G
PF
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M
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LR
WT
C
A
H
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8
Y
C
X
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N
P
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T
M
F
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E
A
D
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L
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H
I
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Y
10
PY
S
11
I
R
V
W
C
P
YL
G
QE
12
L
Y
S
PQ
G
T
R
V
A
W
I
J
F
H
M
N
K
D
13
T
X
I
H
PG
QE
14 15
T
VE
16
E
XR
A
N
P
V
Q
I
M
G
T
Y
K
F
L
W
S
J
C
D
H
17
Q
G
PN
V
J
T
M
K
F
R
L
D
H
W
A
I
17a
G
IF
W
XP
18 19 20
AV
Y
S
GM
C
F
Q
T
K
L
H
E
P
I
X
D
W
J
N
20a
L
X
T
N
K
CG
W
V
PY
S
E
M
J
H
I
R
D
20b
RE
S
21
LC
22
P
X
NSG
Y
23
LR
Q
VY
G
C
T
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24X
25
A
V
P
I
S
L
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RD
26
L
M
H
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27
SYC
M
D
A
J
T
L
I
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H
KN
R
V
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W
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F
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28
X
QD
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M
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S
A
F
L
T
P
R
H
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V
29
I
S
M
L
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R
H
N
V
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T
K
A
30
L
G
V
JP
31
AM
GR
L
T
CH
P
S
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W
32
Y
X
WM
SR
C
I
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P
F
L
A
G
H
T
V
33 34
S
R
G
T
P
V
L
A
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Q
E
35
D
Y
K
Q
H
E
N
36
S
R
J
X
I
K
M
N
T
37 38
G
HR
P
E
A
V
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L
39
G
V
WJ
E
S
P
A
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R
L
T
N
H
C
M
K
F
Y
40
G
P
E
L
T
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QS
41
T
F
A
Y
K
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P
H
V
R
L
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X
42
Q
VNP
RE
K
A
L
F
T
S
H
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C
J
M
I
43
V
P
F
E
G
T
R
S
L
W
K
Q
M
Y
J
N
C
44
PWM
E
VG
K
C
R
Q
S
Y
L
N
J
45
T
XY
K
L
P
A
RN
Q
V
G
C
M
F
46
LS
Q
Y
47
L
S
Y
Q
G
X
R
P
W
T
H
D
V
A
I
K
J
F
N
M
48
A
HC
G
W
R
Q
J
L
49
LR
H
W
C
K
50
E
C
V
I
T
R
L
G
W
P
H
X
N
A
S
K
Q
Y
51
R
K
T
L
J
P
V
I
XN
H
Q
Y
F
52
G
JR
P
HT
53 54 55 56
A
57 58 59
P
J
WH
M
Q
R
L
K
Y
N
T
V
G
F
E
A
C
60
D
A
H
V
W
KT
N
F
L
Q
G
M
R
S
C
Y
X
E
P
I
J
61
GH
R
62
XF
S
E
K
J
H
W
R
M
G
C
A
P
L
I
V
T
63
S
G
AT
Y
J
L
Q
K
V
N
W
P
R
I
X
64
C
QI
N
Y
F
P
L
R
A
K
W
G
V
J
S
T
M
65
Q
Y
IW
H
KF
T
X
S
P
C
L
R
V
A
G
E
D
66
CHV
JR
N
M
P
L
K
X
G
A
S
I
F
E
T
Q
Y
W
67
R
AN
C
G
W
P
X
F
L
S
D
T
Q
K
V
M
Y
68
R
G
T
P
V
N
A
D
Q
H
Y
M
L
C
F
S
69
V
L
G
Q
X
T
CM
K
S
H
W
D
P
70
LIE
N
FMVH
R
Y
S
A
71
S
P
X
M
DR
K
72
R
XSK
V
I
LJ
E
M
H
W
73
L
I
S
T
QG
C
U
C
G
A
Rhizobiales + Rhodobacteriaceae + Caulobacterales +
Hyphomonadaceae (RRCH)
Rickettsiales +Sphingomonadales + Rhodospirialles (RSR)
U
C
G
A
genome
functionally classified tRNAs
X R C E
J E Y
Score each tRNA
sequence for taxon-specific
functional Class-Informative-
Features (CIFs)
Average scores across tRNAs.
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM 0
1
2
3
4
bits
-1
1
C
I
K
JW
2E
K
3
VKR
S
Y
4
I
M
L
R
D
Y
J
S
T
H
P
W
5
K
N
G
D
A
C
V
P
T
H
Y
M
L
Q
F
S
6
K
C
R
S
X
E
F
L
N
T
G
Y
I
P
M
V
7
I
D
H
W
J
P
Y
V
R
X
E
Q
F
G
L
N
T
C
K
S
A
89
X
C
Q
P
G
T
V
R
A
W
I
J
F
H
M
N
K
D
10
SG
Y
L
11X
12
PT
G
SC
13
L
C
Q
1415
K
VH
T
R
Q
J
C
G
W
L
16
T
H
C
V
R
I
L
M
Y
Q
A
S
E
17
L
K
H
M
R
17a
SR
P
1819R
20
T
AR
20aL
20b
RY
21
L
CI
J
H
T
E
S
R
Q
X
Y
W
V
G
K
D
F
N
P
A
M
22
T
GY
Q
S
L
C
23
YQ
S
P
G
T
V
A
W
R
H
I
D
F
M
N
J
K
24
W
D
T
G
Q
LE
25S
26
X
D
L
A
S
HG
W
JI
C
E
R
M
P
Q
T
V
Y
F
N
K
27
A
G
C
J
X
Y
S
L
R
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ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
tRNA Signatures Reveal a Polyphyletic Origin of SAR11
Strains among Alphaproteobacteria
Katherine C. H. Amrine, Wesley D. Swingley
€
, David H. Ardell*
Program in Quantitative and Systems Biology, University of California, Merced, Merced, California, United States of America
Abstract
Molecular phylogenetics and phylogenomics are subject to noise from horizontal gene transfer (HGT) and bias from
convergence in macromolecular compositions. Extensive variation in size, structure and base composition of
alphaproteobacterial genomes has complicated their phylogenomics, sparking controversy over the origins and closest
relatives of the SAR11 strains. SAR11 are highly abundant, cosmopolitan aquatic Alphaproteobacteria with streamlined, A+T-
biased genomes. A dominant view holds that SAR11 are monophyletic and related to both Rickettsiales and the ancestor of
mitochondria. Other studies dispute this, finding evidence of a polyphyletic origin of SAR11 with most strains distantly
related to Rickettsiales. Although careful evolutionary modeling can reduce bias and noise in phylogenomic inference,
entirely different approaches may be useful to extract robust phylogenetic signals from genomes. Here we develop simple
phyloclassifiers from bioinformatically derived tRNA Class-Informative Features (CIFs), features predicted to target tRNAs for
specific interactions within the tRNA interaction network. Our tRNA CIF-based model robustly and accurately classifies
alphaproteobacterial genomes into one of seven undisputed monophyletic orders or families, despite great variability in
tRNA gene complement sizes and base compositions. Our model robustly rejects monophyly of SAR11, classifying all but
one strain as Rhizobiales with strong statistical support. Yet remarkably, conventional phylogenetic analysis of tRNAs
classifies all SAR11 strains identically as Rickettsiales. We attribute this discrepancy to convergence of SAR11 and
Rickettsiales tRNA base compositions. Thus, tRNA CIFs appear more robust to compositional convergence than tRNA
sequences generally. Our results suggest that tRNA-CIF-based phyloclassification is robust to HGT of components of the
tRNA interaction network, such as aminoacyl-tRNA synthetases. We explain why tRNAs are especially advantageous for
prediction of traits governing macromolecular interactions from genomic data, and why such traits may be advantageous in
the search for robust signals to address difficult problems in classification and phylogeny.
Citation:Amrine KCH, Swingley WD, Ardell DH (2014) tRNA Signatures Reveal a Polyphyletic Origin of SAR11 Strains among Alphaproteobacteria. PLoS Comput
Biol 10(2): e1003454. doi:10.1371/journal.pcbi.1003454
Editor:Christos A. Ouzounis, The Centre for Research and Technology, Hellas, Greece
ReceivedMay 1, 2013;AcceptedDecember 10, 2013;PublishedFebruary 27, 2014
Copyright:!2014 Amrine et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:This work was supported by UC Merced. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests:The authors have declared that no competing interests exist.
* E-mail: [email protected]
€ Current address: Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, United States of America.
Introduction
Which parts of genomes are most resistant to compositional
convergence? Which information is vertically inherited most
faithfully? Compositional stationarity and vertical (co-)inheritance
are key, yet frequently violated, assumptions of most current
approaches in molecular phylogenetics and phylogenomics [1].
Horizontal gene transfer (HGT), for example, is so common and
widespread that the very existence of a ‘‘Tree of Life’’ has been
called into question [2,3]. Advances in understanding the history
of life will require discovery of new universal, slowly-evolving
phylogenetic markers that are resistant to compositional conver-
gence and HGT.
The controversial phylogeny ofCa.Pelagibacter ubique
(SAR11) is a case in point. SAR11 make up between a fifth and
a third of the bacterial biomass in marine and freshwater
ecosystems [4]. SAR11 have very small cell sizes, genome sizes,
and intergenic region sizes, possibly in adaptation to extreme
nutrient limitations [5]. Some recent phylogenomic studies place
free-living SAR11 together in a clade with the largely endopar-
asitic Rickettsiales and the alphaproteobacterial ancestor of
mitochondria [6,7,8]. Other studies persuasively argue that this
placement is an artifact of independent convergence of SAR11
and Rickettsiales towards increased genomic A+T contents, and
that SAR11 are more closely related to the free-living Alphaproteo-
bacteria such as the Rhizobiales and Rhodobacteraceae [9,10,11].
The monophyly of SAR11 was also recently rejected [10,12].
Nonstationary macromolecular compositions are a known
source of bias in phylogenomics [13,14]. Widespread variation
in macromolecular compositions may be caused by loss of DNA
repair pathways in reduced genomes [15,11], unveiling an
inherent A+T-bias of mutation in bacteria [16] that elevates
genomic A+T contents [17,18]. A process such as this has likely
altered protein and RNA compositions genome-wide in SAR11,
and if such effects are accounted for, SAR11 appear more closely
related to Rhizobiales and Rhodobacteraceae than Rickettsiales
[10,11]. Consistent with this interpretation, SAR11 strain
HTTC1062 shares, with a large clade of free-living Alphaproteo-
bacteria that excludes the Rickettsiales, a unique and derived
codivergence of features that govern recognition between
tRNAHis and histidyl-tRNA synthetase (HisRS) [19,20]. This
unique functionally significant synapomorphy likely arose only
PLOS Computational Biology | www.ploscompbiol.org 1 February 2014 | Volume 10 | Issue 2 | e1003454
Katie Amrine, Ph.D.
tRNA Signatures Reveal a Polyphyletic Origin of SAR11
Strains among Alphaproteobacteria
Katherine C. H. Amrine, Wesley D. Swingley
€
, David H. Ardell*
Program in Quantitative and Systems Biology, University of California, Merced, Merced, California, United States of America
Abstract
Molecular phylogenetics and phylogenomics are subject to noise from horizontal gene transfer (HGT) and bias from
convergence in macromolecular compositions. Extensive variation in size, structure and base composition of
alphaproteobacterial genomes has complicated their phylogenomics, sparking controversy over the origins and closest
relatives of the SAR11 strains. SAR11 are highly abundant, cosmopolitan aquatic Alphaproteobacteria with streamlined, A+T-
biased genomes. A dominant view holds that SAR11 are monophyletic and related to both Rickettsiales and the ancestor of
mitochondria. Other studies dispute this, finding evidence of a polyphyletic origin of SAR11 with most strains distantly
related to Rickettsiales. Although careful evolutionary modeling can reduce bias and noise in phylogenomic inference,
entirely different approaches may be useful to extract robust phylogenetic signals from genomes. Here we develop simple
phyloclassifiers from bioinformatically derived tRNA Class-Informative Features (CIFs), features predicted to target tRNAs for
specific interactions within the tRNA interaction network. Our tRNA CIF-based model robustly and accurately classifies
alphaproteobacterial genomes into one of seven undisputed monophyletic orders or families, despite great variability in
tRNA gene complement sizes and base compositions. Our model robustly rejects monophyly of SAR11, classifying all but
one strain as Rhizobiales with strong statistical support. Yet remarkably, conventional phylogenetic analysis of tRNAs
classifies all SAR11 strains identically as Rickettsiales. We attribute this discrepancy to convergence of SAR11 and
Rickettsiales tRNA base compositions. Thus, tRNA CIFs appear more robust to compositional convergence than tRNA
sequences generally. Our results suggest that tRNA-CIF-based phyloclassification is robust to HGT of components of the
tRNA interaction network, such as aminoacyl-tRNA synthetases. We explain why tRNAs are especially advantageous for
prediction of traits governing macromolecular interactions from genomic data, and why such traits may be advantageous in
the search for robust signals to address difficult problems in classification and phylogeny.
Citation:Amrine KCH, Swingley WD, Ardell DH (2014) tRNA Signatures Reveal a Polyphyletic Origin of SAR11 Strains among Alphaproteobacteria. PLoS Comput
Biol 10(2): e1003454. doi:10.1371/journal.pcbi.1003454
Editor:Christos A. Ouzounis, The Centre for Research and Technology, Hellas, Greece
ReceivedMay 1, 2013;AcceptedDecember 10, 2013;PublishedFebruary 27, 2014
Copyright:!2014 Amrine et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:This work was supported by UC Merced. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests:The authors have declared that no competing interests exist.
* E-mail: [email protected]
€ Current address: Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, United States of America.
Introduction
Which parts of genomes are most resistant to compositional
convergence? Which information is vertically inherited most
faithfully? Compositional stationarity and vertical (co-)inheritance
are key, yet frequently violated, assumptions of most current
approaches in molecular phylogenetics and phylogenomics [1].
Horizontal gene transfer (HGT), for example, is so common and
widespread that the very existence of a ‘‘Tree of Life’’ has been
called into question [2,3]. Advances in understanding the history
of life will require discovery of new universal, slowly-evolving
phylogenetic markers that are resistant to compositional conver-
gence and HGT.
The controversial phylogeny ofCa.Pelagibacter ubique
(SAR11) is a case in point. SAR11 make up between a fifth and
a third of the bacterial biomass in marine and freshwater
ecosystems [4]. SAR11 have very small cell sizes, genome sizes,
and intergenic region sizes, possibly in adaptation to extreme
nutrient limitations [5]. Some recent phylogenomic studies place
free-living SAR11 together in a clade with the largely endopar-
asitic Rickettsiales and the alphaproteobacterial ancestor of
mitochondria [6,7,8]. Other studies persuasively argue that this
placement is an artifact of independent convergence of SAR11
and Rickettsiales towards increased genomic A+T contents, and
that SAR11 are more closely related to the free-living Alphaproteo-
bacteria such as the Rhizobiales and Rhodobacteraceae [9,10,11].
The monophyly of SAR11 was also recently rejected [10,12].
Nonstationary macromolecular compositions are a known
source of bias in phylogenomics [13,14]. Widespread variation
in macromolecular compositions may be caused by loss of DNA
repair pathways in reduced genomes [15,11], unveiling an
inherent A+T-bias of mutation in bacteria [16] that elevates
genomic A+T contents [17,18]. A process such as this has likely
altered protein and RNA compositions genome-wide in SAR11,
and if such effects are accounted for, SAR11 appear more closely
related to Rhizobiales and Rhodobacteraceae than Rickettsiales
[10,11]. Consistent with this interpretation, SAR11 strain
HTTC1062 shares, with a large clade of free-living Alphaproteo-
bacteria that excludes the Rickettsiales, a unique and derived
codivergence of features that govern recognition between
tRNAHis and histidyl-tRNA synthetase (HisRS) [19,20]. This
unique functionally significant synapomorphy likely arose only
PLOS Computational Biology | www.ploscompbiol.org 1 February 2014 | Volume 10 | Issue 2 | e1003454
ACC STEM
U73 conveys
about six times
more information
about tRNA
function than A73
U73 is equally over-
represented in tRNA
Cys
and tRNA
Gly
8597 tRNA genes from 147 alphaproteobacterial genomes
The height of a letter
quantifies both
functional information
of a CIF and its
relative over-
representation in a
particular functional
class of tRNAs.
Function logos visualize information about
function, conditioned by a common structure
Visualizing bacterial tRNA identity determinants and
antideterminants using function logos and inverse
function logos
Eva Freyhult, Vincent Moulton
1
and David H. Ardell*
Linnaeus Centre for Bioinformatics, Uppsala University, Uppsala, Sweden and
1
School of Computing Sciences,
University of East Anglia, Norwich NR4 7TJ, UK
Received November 21, 2005; Revised and Accepted January 12, 2006
ABSTRACT
Sequence logos are stacked bar graphs that gen-
eralize the notion of consensus sequence. They
employ entropy statistics very effectively to display
variation in a structural alignment of sequences
of a common function, while emphasizing its over-
represented features. Yet sequence logos cannot
display features that distinguish functional sub-
classes within a structurally related superfamily
nor do they display under-represented features. We
introduce two extensions to address these needs:
function logos and inverse logos. Function logos
display subfunctions that are over-represented
among sequences carrying a specific feature.
Inverse logos generalize both sequence logos and
function logos by displaying under-represented,
rather than over-represented, features or functions
in structural alignments. To make inverse logos,
a compositional inverse is applied to the feature
or function frequency distributions before logo
construction, where a compositional inverse is a
mathematical transform that makes common fea-
tures or functions rare andvice versa. We applied
these methods to a database of structurally aligned
bacterial tDNAs to create highly condensed, birds-
eye views of potentially all so-called identity
determinants and antideterminants that confer spe-
cific amino acid charging or initiator function on
tRNAs in bacteria. We recovered both known
and a few potentially novel identity elements.
Function logos and inverse logos are useful tools
for exploratory bioinformatic analysis of structure–
function relationships in sequence families and
superfamilies.
INTRODUCTION
Which sequence features confer a specific biological function
on a class of macromolecules? This question becomes both
more acute and more tractable when contrasting classes of
molecules with distinct functions yet highly similar structures.
In that situation, there may be fewer structural differences
to explain the functional differences among classes, but this
also means fewer structural differences to detect and test.
Highly similar structures also make structural analogy easier
to assign. A common complication arises, however, in that
structural similarity may derive from common ancestry rather
than from functional constraint.
An example of such a problem is transfer RNA (tRNA)
identity [reviewed in (1–4)]. A pillar of fidelity in gene exp-
ression is the consistency with which specific amino acids are
attached to specific tRNAs by enzymes called aminoacyl-
tRNA synthetases (aaRSs). In general, there is one population
of aaRS in a cell for each of the 20 canonical amino acids.
Despite the generally very high structural similarity of all
tRNAs, each must interact productively with only one syn-
thetase population to be charged with its cognate amino acid
and interact nonproductively with the remaining 19 enzyme
populations. Theidentityof a tRNA refers to this amino acid
‘charging’ specificity. In a simplification, the identity of a
tRNA can be thought to depend on a constellation of structural
features called ‘identity elements,’ encompassing features that
promote recognition and catalytic activity by its cognate aaRS
(called ‘determinants’) or discrimination by noncognate
synthetases against the same or other features (called ‘anti-
determinants’) so as to inhibit translationally ambiguous
tRNA-binding and aminoacylation.
We (roughly) define a ‘tRNA identity code’ as the set of all
identity elements that make tRNAs functionally distinct within
a taxonomic lineage. We note five points about identity codes
as described in more detail in the aforementioned reviews.
First, a complete identity code has never been completely
described for any taxonomic lineage. Second, identity codes
*To whom correspondence should be addressed at David Ardell, Linnaeus Centre for Bioinformatics, Box 598, 751 24 Uppsala, Sweden. Tel: +46 18 471 6694;
Fax: +46 18 471 6698; E-mail: [email protected]
!The Author 2006. Published by Oxford University Press. All rights reserved.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access
version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and OxfordUniversity Press
are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but
only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected]
Nucleic Acids Research, 2006, Vol. 34, No. 3905–916
doi:10.1093/nar/gkj478
Published online February 9, 2006
Eva Freyhult, Ph.D.
1
2
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L
E
P
N
V
J
X
32 33 34
L
A
P
T
V
G
S
Y
C
H
I
N
D
F
35
S
A
P
T
36
R
L
H
Q
P
37
GXI
R
FQ
L
P
H
38
LG
AP
39
FQP
L
V
T
S
R
E
A
G
D
I
40
D
QMI
A
L
P
R
S
G
J
E
T
V
41
EKL
P
M
R
V
F
T
S
N
J
I
42
I
J
M
V
N
H
L
TG
F
C
S
A
R
P
Y
K
Q
E
X
D
W
43
N
F
J
Y
X
Q
KW
M
G
R
E
P
L
T
S
V
A
H
I
D
44
LNH
E
Q
A
I
D
R
T
W
G
V
K
M
P
C
F
45
C
F
Q
S
E
L
Y
M
G
V
N
R
A
P
K
W
I
X
T
H
D
J
46
S
E
Y
Q
G
L
H
R
T
D
V
X
W
A
K
J
I
M
F
P
N
47
R
ELQ
48
CY
E
49
E
CQ
V
AK
R
H
Y
X
T
S
F
G
J
W
M
L
P
I
D
N
50
K
N
XC
G
Q
S
P
W
H
R
V
T
J
L
A
F
E
M
I
51
I
N
H
F
XC
A
R
P
V
L
K
J
T
W
M
E
G
D
S
52 53 54E
55 56 57
X
I
M
H
V
T
YJ
P
C
L
F
W
S
R
G
D
K
E
A
Q
N
58K
59
A
GC
Q
E
P
R
N
Y
V
K
T
M
H
W
L
J
I
D
6061 62
L
TG
I
R
H
63
JX
Y
P
G
I
E
K
V
M
T
L
H
N
A
C
F
64
M
SE
F
K
T
L
G
Y
H
J
W
V
R
P
N
Q
X
C
D
65
L
X
P
W
G
R
M
V
F
J
T
S
Y
H
K
C
Q
66
G
L
RVY
67
X
MD
S
I
V
N
T
Q
L
G
K
P
C
H
W
A
E
R
J
68
G
F
M
SV
C
L
P
K
H
N
T
A
J
R
E
I
69
Q
G
MST
Y
L
N
I
K
C
V
F
J
R
E
70
S
P
QJE
R
L
H
T
M
F
N
C
X
71
V
Q
J
W
KR
E
D
L
H
F
N
G
T
72
V
R
N
FP
73
X
T
FE
R
Q
N
S
D
W 0
1
2
3
4
bits
-1
1
L
IXN
Q
2
LK
H
E
R
3
V
L
XM
SR
E
T
4
X
G
L
P
J
T
SR
F
M
N
V
K
I
5
EV
P
QA
M
J
RT
N
L
I
D
C
H
6
Y
MR
S
CK
V
Q
A
L
H
I
P
T
G
D
J
W
7
G
PF
Q
M
S
LR
WT
C
A
H
K
V
E
8
Y
C
X
Q
R
N
P
VW
S
T
M
F
G
E
A
D
K
L
J
H
I
9G
Y
10
PY
S
11
I
R
V
W
C
P
YL
G
QE
12
L
Y
S
PQ
G
T
R
V
A
W
I
J
F
H
M
N
K
D
13
T
X
I
H
PG
QE
14 15
T
VE
16
E
XR
A
N
P
V
Q
I
M
G
T
Y
K
F
L
W
S
J
C
D
H
17
Q
G
PN
V
J
T
M
K
F
R
L
D
H
W
A
I
17a
G
IF
W
XP
18 19 20
AV
Y
S
GM
C
F
Q
T
K
L
H
E
P
I
X
D
W
J
N
20a
L
X
T
N
K
CG
W
V
PY
S
E
M
J
H
I
R
D
20b
RE
S
21
LC
22
P
X
NSG
Y
23
LR
Q
VY
G
C
T
S
24X
25
A
V
P
I
S
L
G
RD
26
L
M
H
S
J
Q
27
SYC
M
D
A
J
T
L
I
Q
H
KN
R
V
E
W
P
F
GX
28
X
QD
J
E
K
M
G
S
A
F
L
T
P
R
H
N
V
29
I
S
M
L
XG
QY
R
H
N
V
P
T
K
A
30
L
G
V
JP
31
AM
GR
L
T
CH
P
S
Q
W
32
Y
X
WM
SR
C
I
E
Q
P
F
L
A
G
H
T
V
33 34
S
R
G
T
P
V
L
A
K
Q
E
35
D
Y
K
Q
H
E
N
36
S
R
J
X
I
K
M
N
T
37 38
G
HR
P
E
A
V
Q
L
39
G
V
WJ
E
S
P
A
Q
R
L
T
N
H
C
M
K
F
Y
40
G
P
E
L
T
R
QS
41
T
F
A
Y
K
Q
P
H
V
R
L
E
G
X
42
Q
VNP
RE
K
A
L
F
T
S
H
G
C
J
M
I
43
V
P
F
E
G
T
R
S
L
W
K
Q
M
Y
J
N
C
44
PWM
E
VG
K
C
R
Q
S
Y
L
N
J
45
T
XY
K
L
P
A
RN
Q
V
G
C
M
F
46
LS
Q
Y
47
L
S
Y
Q
G
X
R
P
W
T
H
D
V
A
I
K
J
F
N
M
48
A
HC
G
W
R
Q
J
L
49
LR
H
W
C
K
50
E
C
V
I
T
R
L
G
W
P
H
X
N
A
S
K
Q
Y
51
R
K
T
L
J
P
V
I
XN
H
Q
Y
F
52
G
JR
P
HT
53 54 55 56
A
57 58 59
P
J
WH
M
Q
R
L
K
Y
N
T
V
G
F
E
A
C
60
D
A
H
V
W
KT
N
F
L
Q
G
M
R
S
C
Y
X
E
P
I
J
61
GH
R
62
XF
S
E
K
J
H
W
R
M
G
C
A
P
L
I
V
T
63
S
G
AT
Y
J
L
Q
K
V
N
W
P
R
I
X
64
C
QI
N
Y
F
P
L
R
A
K
W
G
V
J
S
T
M
65
Q
Y
IW
H
KF
T
X
S
P
C
L
R
V
A
G
E
D
66
CHV
JR
N
M
P
L
K
X
G
A
S
I
F
E
T
Q
Y
W
67
R
AN
C
G
W
P
X
F
L
S
D
T
Q
K
V
M
Y
68
R
G
T
P
V
N
A
D
Q
H
Y
M
L
C
F
S
69
V
L
G
Q
X
T
CM
K
S
H
W
D
P
70
LIE
N
FMVH
R
Y
S
A
71
S
P
X
M
DR
K
72
R
XSK
V
I
LJ
E
M
H
W
73
L
I
S
T
QG
C 0
1
2
3
4
bits
-1
1
L
V
R
NX
P
2
V
Q
J
W
KR
E
D
L
H
F
N
G
T
3
S
Q
PJR
E
L
H
T
M
F
N
X
C
4
G
Q
MST
Y
L
N
I
K
C
V
F
J
R
E
5
G
Y
FS
V
M
C
L
P
K
H
N
T
I
A
R
J
E
6
X
MW
D
JS
I
T
V
G
N
Q
L
P
K
C
H
A
E
R
7
G
L
RVY
89
GQE
10
SY
11
XQ
GL
Y
P
C
W
V
R
I
S
M
K
F
A
T
J
H
N
D
12
LQ
S
Y
E
13
L
E
QG
P
H
X
I
T
D
A
W
V
K
J
F
R
N
M
14Y
15
L
G
T
H
16
C
I
G
F
H
D
Y
S
J
W
L
T
K
M
Q
V
P
N
A
R
X
E
17
S
VN
AL
M
T
RD
KP
X
17a
SPX
18 19 20
V
X
P
E
H
K
L
T
Q
C
M
GS
Y
20a
L
R
P
I
V
X
YJ
M
WE
S
20b
LE
21 22Y
23
RS
Q
GP
C
L
X
24
SX
Y
Q
G
25
D
YR
S
G
L
I
P
V
A
C
E
T
W
X
F
K
J
M
Q
H
N
26
PLQ
GJ
27
F
Y
X
N
C
Q
K
W
M
G
R
E
P
T
L
V
S
A
H
I
D
28
I
J
M
V
N
H
L
TG
F
C
S
A
R
P
Y
K
Q
E
D
X
W
29
G
EKL
P
M
R
V
F
T
S
N
J
I
30
D
Q
MI
A
L
P
R
S
G
J
E
T
V
31
FQP
L
V
T
S
R
E
A
G
D
I
32
V
A
TH
M
G
L
P
F
Q
E
I
C
R
S
W
X
Y
K
J
N
D
33 34
A
S
G
V
E
P
T
Q
L
K
R
X
J
M
W
35
SC
W
G
R
36
D
A
G
E
V
37 38
I
G
XL
R
A
S
Y
H
Q
E
V
D
39
G
R
Q
H
K
AM
L
E
P
N
V
J
X
40
V
T
E
J
S
G
R
P
L
A
I
M
Q
D
F
W
X
Y
K
H
N
C
41
X
G
VAP
T
J
R
E
H
S
L
F
Y
M
Q
C
D
W
42
SI
P
R
K
MG
Q
A
T
N
L
Y
F
J
43
I
T
V
XCS
A
P
GL
E
R
W
N
M
J
Y
K
Q
F
44
E
K
H
J
C
W
R
N
GL
Y
S
Q
45
P
Q
YGC
46
Q
Y
L
S
47
G
K
H
I
A
S
V
D
T
W
P
Q
R
Y
X
48
L
JE
Q
Y
R
G
W
C
H
T
V
X
A
S
I
F
D
K
P
N
M
49
L
X
P
W
G
R
M
V
F
J
T
S
Y
H
K
C
Q
50
M
SE
F
K
T
L
G
Q
P
Y
H
J
W
R
V
N
X
C
D
51
J
X
YP
G
I
K
E
F
V
M
N
T
L
H
A
C
52
L
M
TG
I
R
H
53 54 55
T
56 57 58 59
K
V
X
N
I
G
R
C
T
F
A
LH
E
60
SR
M
G
Q
L
FN
T
K
H
W
V
A
D
61 62 63
I
N
F
XC
H
R
A
P
V
L
W
K
J
T
M
E
G
S
D
64
V
K
XS
C
W
G
Q
T
R
M
P
H
L
A
F
E
I
65
E
CG
Q
V
A
K
R
H
L
S
P
Y
T
X
F
J
W
M
I
N
66
Y
T
W
V
AK
Q
F
I
H
S
C
G
X
R
L
M
P
N
J
D
67
G
A
J
R
T
K
Q
E
L
V
C
I
M
Y
N
S
X
F
68
R
A
J
H
C
TS
N
L
F
D
M
K
P
Y
V
Q
G
X
W
69
R
V
FJC
P
N
D
L
Y
W
T
H
S
M
Q
X
G
A
70
M
SRH
Y
E
L
J
V
Q
P
I
G
D
K
W
71
T
G
N
L
F
D
RE
K
M
W
J
Q
I
X
V
P
S
Y
C
A
72
P
HM
EJ
L
I
V
K
S
F
R
C
A
Y
D
T
G
73
A
P
N
HX
U
C
G
A
Rhizobiales + Rhodobacteriaceae + Caulobacterales +
Hyphomonadaceae (RRCH)
Rickettsiales +Sphingomonadales + Rhodospirialles (RSR)
U
C
G
A
genome
functionally classified tRNAs
X R C E
J E Y
Score each tRNA
sequence for taxon-specific
functional Class-Informative-
Features (CIFs)
Average scores across tRNAs.
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM 0
1
2
3
4
bits
-1
1
C
I
K
JW
2E
K
3
VKR
S
Y
4
I
M
L
R
D
Y
J
S
T
H
P
W
5
K
N
G
D
A
C
V
P
T
H
Y
M
L
Q
F
S
6
K
C
R
S
X
E
F
L
N
T
G
Y
I
P
M
V
7
I
D
H
W
J
P
Y
V
R
X
E
Q
F
G
L
N
T
C
K
S
A
89
X
C
Q
P
G
T
V
R
A
W
I
J
F
H
M
N
K
D
10
SG
Y
L
11X
12
PT
G
SC
13
L
C
Q
1415
K
VH
T
R
Q
J
C
G
W
L
16
T
H
C
V
R
I
L
M
Y
Q
A
S
E
17
L
K
H
M
R
17a
SR
P
1819R
20
T
AR
20aL
20b
RY
21
L
CI
J
H
T
E
S
R
Q
X
Y
W
V
G
K
D
F
N
P
A
M
22
T
GY
Q
S
L
C
23
YQ
S
P
G
T
V
A
W
R
H
I
D
F
M
N
J
K
24
W
D
T
G
Q
LE
25S
26
X
D
L
A
S
HG
W
JI
C
E
R
M
P
Q
T
V
Y
F
N
K
27
A
G
C
J
X
Y
S
L
R
T
M
K
F
Q
N
28
A
M
V
J
R
T
D
S
G
N
L
C
F
I
29
K
TA
D
S
W
V
P
M
R
L
E
H
Y
X
G
30
S
T
31
VH
SP
N
Q
A
R
L
T
M
K
C
Y
F
32
AM
3334R
35
X
V
I
F
J
M
L
36
L
S
C
Y
F
W
37
LQ
R
V
S
E
Y
A
X
T
W
DI
F
G
J
M
N
K
C
38
D
HL
AP
T
G
S
R
Y
W
X
F
K
J
M
C
I
N
39
VI
A
R
T
LP
H
Q
S
W
40
T
41
L
S
HF
R
J
D
N
P
T
V
A
K
42
H
I
D
S
M
W
J
G
T
A
L
N
R
K
P
V
43
LQJ
N
S
T
E
G
K
F
P
D
W
A
V
R
H
X
44
L
SM
C
N
K
P
J
G
E
V
H
W
R
A
I
T
D
X
45
R
A
W
P
T
M
N
L
J
V
S
G
C
Q
E
46
S
T
W
Y
Q
L
G
C
E
47
L
Q
DIY
48
G
A
V
R
TD
49
IL
G
F
P
Y
J
S
K
H
X
T
W
C
R
V
A
E
50
RL
I
W
V
X
T
Q
K
A
Y
S
G
51
AT
W
N
K
V
L
P
R
X
Y
J
Q
I
52
S
QG
A
W
K
Y
RD
P
V
H
L
T
I
5354555657
SG
AK
H
E
P
R
W
C
D
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C
D
H
17
Q
G
PN
V
J
T
M
K
F
R
L
D
H
W
A
I
17a
G
IF
W
XP
18 19 20
AV
Y
S
GM
C
F
Q
T
K
L
H
E
P
I
X
D
W
J
N
20a
L
X
T
N
K
CG
W
V
PY
S
E
M
J
H
I
R
D
20b
RE
S
21
LC
22
P
X
NSG
Y
23
LR
Q
VY
G
C
T
S
24X
25
A
V
P
I
S
L
G
RD
26
L
M
H
S
J
Q
27
SYC
M
D
A
J
T
L
I
Q
H
KN
R
V
E
W
P
F
GX
28
X
QD
J
E
K
M
G
S
A
F
L
T
P
R
H
N
V
29
I
S
M
L
XG
QY
R
H
N
V
P
T
K
A
30
L
G
V
JP
31
AM
GR
L
T
CH
P
S
Q
W
32
Y
X
WM
SR
C
I
E
Q
P
F
L
A
G
H
T
V
33 34
S
R
G
T
P
V
L
A
K
Q
E
35
D
Y
K
Q
H
E
N
36
S
R
J
X
I
K
M
N
T
37 38
G
HR
P
E
A
V
Q
L
39
G
V
WJ
E
S
P
A
Q
R
L
T
N
H
C
M
K
F
Y
40
G
P
E
L
T
R
QS
41
T
F
A
Y
K
Q
P
H
V
R
L
E
G
X
42
Q
VNP
RE
K
A
L
F
T
S
H
G
C
J
M
I
43
V
P
F
E
G
T
R
S
L
W
K
Q
M
Y
J
N
C
44
PWM
E
VG
K
C
R
Q
S
Y
L
N
J
45
T
XY
K
L
P
A
RN
Q
V
G
C
M
F
46
LS
Q
Y
47
L
S
Y
Q
G
X
R
P
W
T
H
D
V
A
I
K
J
F
N
M
48
A
HC
G
W
R
Q
J
L
49
LR
H
W
C
K
50
E
C
V
I
T
R
L
G
W
P
H
X
N
A
S
K
Q
Y
51
R
K
T
L
J
P
V
I
XN
H
Q
Y
F
52
G
JR
P
HT
53 54 55 56
A
57 58 59
P
J
WH
M
Q
R
L
K
Y
N
T
V
G
F
E
A
C
60
D
A
H
V
W
KT
N
F
L
Q
G
M
R
S
C
Y
X
E
P
I
J
61
GH
R
62
XF
S
E
K
J
H
W
R
M
G
C
A
P
L
I
V
T
63
S
G
AT
Y
J
L
Q
K
V
N
W
P
R
I
X
64
C
QI
N
Y
F
P
L
R
A
K
W
G
V
J
S
T
M
65
Q
Y
IW
H
KF
T
X
S
P
C
L
R
V
A
G
E
D
66
CHV
JR
N
M
P
L
K
X
G
A
S
I
F
E
T
Q
Y
W
67
R
AN
C
G
W
P
X
F
L
S
D
T
Q
K
V
M
Y
68
R
G
T
P
V
N
A
D
Q
H
Y
M
L
C
F
S
69
V
L
G
Q
X
T
CM
K
S
H
W
D
P
70
LIE
N
FMVH
R
Y
S
A
71
S
P
X
M
DR
K
72
R
XSK
V
I
LJ
E
M
H
W
73
L
I
S
T
QG
C 0
1
2
3
4
bits
-1
1
L
V
R
NX
P
2
V
Q
J
W
KR
E
D
L
H
F
N
G
T
3
S
Q
PJR
E
L
H
T
M
F
N
X
C
4
G
Q
MST
Y
L
N
I
K
C
V
F
J
R
E
5
G
Y
FS
V
M
C
L
P
K
H
N
T
I
A
R
J
E
6
X
MW
D
JS
I
T
V
G
N
Q
L
P
K
C
H
A
E
R
7
G
L
RVY
89
GQE
10
SY
11
XQ
GL
Y
P
C
W
V
R
I
S
M
K
F
A
T
J
H
N
D
12
LQ
S
Y
E
13
L
E
QG
P
H
X
I
T
D
A
W
V
K
J
F
R
N
M
14Y
15
L
G
T
H
16
C
I
G
F
H
D
Y
S
J
W
L
T
K
M
Q
V
P
N
A
R
X
E
17
S
VN
AL
M
T
RD
KP
X
17a
SPX
18 19 20
V
X
P
E
H
K
L
T
Q
C
M
GS
Y
20a
L
R
P
I
V
X
YJ
M
WE
S
20b
LE
21 22Y
23
RS
Q
GP
C
L
X
24
SX
Y
Q
G
25
D
YR
S
G
L
I
P
V
A
C
E
T
W
X
F
K
J
M
Q
H
N
26
PLQ
GJ
27
F
Y
X
N
C
Q
K
W
M
G
R
E
P
T
L
V
S
A
H
I
D
28
I
J
M
V
N
H
L
TG
F
C
S
A
R
P
Y
K
Q
E
D
X
W
29
G
EKL
P
M
R
V
F
T
S
N
J
I
30
D
Q
MI
A
L
P
R
S
G
J
E
T
V
31
FQP
L
V
T
S
R
E
A
G
D
I
32
V
A
TH
M
G
L
P
F
Q
E
I
C
R
S
W
X
Y
K
J
N
D
33 34
A
S
G
V
E
P
T
Q
L
K
R
X
J
M
W
35
SC
W
G
R
36
D
A
G
E
V
37 38
I
G
XL
R
A
S
Y
H
Q
E
V
D
39
G
R
Q
H
K
AM
L
E
P
N
V
J
X
40
V
T
E
J
S
G
R
P
L
A
I
M
Q
D
F
W
X
Y
K
H
N
C
41
X
G
VAP
T
J
R
E
H
S
L
F
Y
M
Q
C
D
W
42
SI
P
R
K
MG
Q
A
T
N
L
Y
F
J
43
I
T
V
XCS
A
P
GL
E
R
W
N
M
J
Y
K
Q
F
44
E
K
H
J
C
W
R
N
GL
Y
S
Q
45
P
Q
YGC
46
Q
Y
L
S
47
G
K
H
I
A
S
V
D
T
W
P
Q
R
Y
X
48
L
JE
Q
Y
R
G
W
C
H
T
V
X
A
S
I
F
D
K
P
N
M
49
L
X
P
W
G
R
M
V
F
J
T
S
Y
H
K
C
Q
50
M
SE
F
K
T
L
G
Q
P
Y
H
J
W
R
V
N
X
C
D
51
J
X
YP
G
I
K
E
F
V
M
N
T
L
H
A
C
52
L
M
TG
I
R
H
53 54 55
T
56 57 58 59
K
V
X
N
I
G
R
C
T
F
A
LH
E
60
SR
M
G
Q
L
FN
T
K
H
W
V
A
D
61 62 63
I
N
F
XC
H
R
A
P
V
L
W
K
J
T
M
E
G
S
D
64
V
K
XS
C
W
G
Q
T
R
M
P
H
L
A
F
E
I
65
E
CG
Q
V
A
K
R
H
L
S
P
Y
T
X
F
J
W
M
I
N
66
Y
T
W
V
AK
Q
F
I
H
S
C
G
X
R
L
M
P
N
J
D
67
G
A
J
R
T
K
Q
E
L
V
C
I
M
Y
N
S
X
F
68
R
A
J
H
C
TS
N
L
F
D
M
K
P
Y
V
Q
G
X
W
69
R
V
FJC
P
N
D
L
Y
W
T
H
S
M
Q
X
G
A
70
M
SRH
Y
E
L
J
V
Q
P
I
G
D
K
W
71
T
G
N
L
F
D
RE
K
M
W
J
Q
I
X
V
P
S
Y
C
A
72
P
HM
EJ
L
I
V
K
S
F
R
C
A
Y
D
T
G
73
A
P
N
HX
U
C
G
A
Rhizobiales + Rhodobacteriaceae + Caulobacterales +
Hyphomonadaceae (RRCH)
Rickettsiales +Sphingomonadales + Rhodospirialles (RSR)
U
C
G
A
genome
functionally classified tRNAs
X R C E
J E Y
Score each tRNA
sequence for taxon-specific
functional Class-Informative-
Features (CIFs)
Average scores across tRNAs.
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM 0
1
2
3
4
bits
-1
1
C
I
K
JW
2E
K
3
VKR
S
Y
4
I
M
L
R
D
Y
J
S
T
H
P
W
5
K
N
G
D
A
C
V
P
T
H
Y
M
L
Q
F
S
6
K
C
R
S
X
E
F
L
N
T
G
Y
I
P
M
V
7
I
D
H
W
J
P
Y
V
R
X
E
Q
F
G
L
N
T
C
K
S
A
89
X
C
Q
P
G
T
V
R
A
W
I
J
F
H
M
N
K
D
10
SG
Y
L
11X
12
PT
G
SC
13
L
C
Q
1415
K
VH
T
R
Q
J
C
G
W
L
16
T
H
C
V
R
I
L
M
Y
Q
A
S
E
17
L
K
H
M
R
17a
SR
P
1819R
20
T
AR
20aL
20b
RY
21
L
CI
J
H
T
E
S
R
Q
X
Y
W
V
G
K
D
F
N
P
A
M
22
T
GY
Q
S
L
C
23
YQ
S
P
G
T
V
A
W
R
H
I
D
F
M
N
J
K
24
W
D
T
G
Q
LE
25S
26
X
D
L
A
S
HG
W
JI
C
E
R
M
P
Q
T
V
Y
F
N
K
27
A
G
C
J
X
Y
S
L
R
T
M
K
F
Q
N
28
A
M
V
J
R
T
D
S
G
N
L
C
F
I
29
K
TA
D
S
W
V
P
M
R
L
E
H
Y
X
G
30
S
T
31
VH
SP
N
Q
A
R
L
T
M
K
C
Y
F
32
AM
3334R
35
X
V
I
F
J
M
L
36
L
S
C
Y
F
W
37
LQ
R
V
S
E
Y
A
X
T
W
DI
F
G
J
M
N
K
C
38
D
HL
AP
T
G
S
R
Y
W
X
F
K
J
M
C
I
N
39
VI
A
R
T
LP
H
Q
S
W
40
T
41
L
S
HF
R
J
D
N
P
T
V
A
K
42
H
I
D
S
M
W
J
G
T
A
L
N
R
K
P
V
43
LQJ
N
S
T
E
G
K
F
P
D
W
A
V
R
H
X
44
L
SM
C
N
K
P
J
G
E
V
H
W
R
A
I
T
D
X
45
R
A
W
P
T
M
N
L
J
V
S
G
C
Q
E
46
S
T
W
Y
Q
L
G
C
E
47
L
Q
DIY
48
G
A
V
R
TD
49
IL
G
F
P
Y
J
S
K
H
X
T
W
C
R
V
A
E
50
RL
I
W
V
X
T
Q
K
A
Y
S
G
51
AT
W
N
K
V
L
P
R
X
Y
J
Q
I
52
S
QG
A
W
K
Y
RD
P
V
H
L
T
I
5354555657
SG
AK
H
E
P
R
W
C
D
Y
F
V
T
L
J
M
I
X
5859
CL
F
W
A
I
J
V
E
H
T
G
N
K
M
R
Q
X
P
Y
S
60
JHW
6162
M
R
N
T
K
63
T
LP
I
K
R
Q
V
N
X
Y
F
64
VM
Q
L
I
P
D
H
X
A
R
W
T
C
K
G
N
Y
F
S
J
65
Q
T
66
S
LK
A
R
T
V
E
67
M
F
K
V
E
R
A
P
Q
L
W
H
T
J
I
G
D
68
S
A
NJ
T
H
R
K
L
F
I
69
TN
C
R
K
S
V
F
E
I
M
70
S
RE
C
T
K
71
TR
E
72
X
N
Q
73
S
N
QR
E
T
F
Y
X
V
J
I
A
K
P
M
L
0
1
2
3
4
bits
-1
1
X
Q
N
2
TL
R
E
3
S
G
RE
C
T
K
4
L
TK
R
S
E
F
V
M
I
N
C
5
SJ
A
T
N
H
R
K
L
F
I
6
V
F
E
M
R
K
P
Q
A
L
T
H
I
G
J
W
D
7
S
LK
A
R
T
V
E
89
10S
11
S
V
W
D
M
T
Y
G
Q
LE
12
YQ
S
P
G
T
V
A
W
R
H
I
D
F
M
N
J
K
13
L
SV
D
R
T
N
G
QE
1415
A
R
D
C
V
16
P
I
E
X
M
R
A
Q
V
S
T
N
G
L
W
J
K
Y
H
F
D
C
17
PG
N
X
T
K
F
J
L
V
M
R
A
H
W
I
D
17a
R
L
D
W
XP
181920
A
SC
E
W
Y
V
N
K
T
M
L
F
G
D
X
H
Q
P
I
J
20a
TX
P
W
V
C
E
Y
M
H
S
J
R
I
D
20b
R
C
PE
S
21
LTC
22
M
SY
23
PT
G
SC
24X
25
C
SW
M
T
N
I
P
Y
A
V
R
D
L
G
26
A
P
R
C
N
W
LJ
Q
G
27
L
S
QT
N
G
K
E
F
D
W
A
P
R
V
H
X
28
H
I
D
M
W
J
S
T
L
G
N
C
K
A
P
R
V
29
LH
S
F
R
J
D
N
P
V
T
A
K
30
T
LV
P
31
V
GD
I
R
T
L
A
P
H
Q
S
W
32
FR
I
S
G
L
A
P
T
Q
V
H
3334
IR
S
T
G
V
L
P
A
K
Q
E
35
E
H
K
Y
N
Q
D
36
S
R
J
X
I
K
M
N
T
3738
GTPA
V
L
Q
H
39
I
V
S
PN
Q
H
A
R
L
T
C
M
Y
K
F
40
R
L
E
V
H
T
MD
S
Q
41
T
K
A
D
S
W
V
P
M
R
L
E
H
Y
X
G
42
K
H
V
M
R
E
T
A
D
S
N
L
G
J
F
C
I
43
A
X
Y
R
L
K
S
T
J
C
Q
F
M
N
44
T
AI
V
P
W
R
Q
K
H
Y
G
M
E
N
C
S
J
L
45
TA
J
R
P
Q
W
L
N
S
V
K
H
G
C
M
F
46
L
SC
Q
Y
47
S
L
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ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM 0
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ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
0
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H
K
Q
F
N
31
I
K
A
M
Q
L
H
P
V
N
X
J
323334
L
P
A
T
G
V
S
I
Y
C
N
H
D
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35
S
A
P
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36
R
L
H
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P
37
SV
R
Q
L
H
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38
ALP
39
M
C
QL
P
T
S
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R
A
I
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G
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40
S
IA
L
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G
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41
HR
L
V
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K
T
F
S
N
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42
F
JN
C
L
G
V
M
R
P
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A
K
S
D
E
W
H
Y
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43
M
NY
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F
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A
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W
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L
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44
J
QD
L
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T
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A
C
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H
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45
C
S
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M
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H
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K
N
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W
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A
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46
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L
G
W
T
R
D
A
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V
F
J
I
H
K
P
N
M
47
LQ
48
A
C
V
G
49
E
A
K
T
V
R
C
Q
W
X
P
H
S
J
Y
F
G
L
I
D
M
N
50
K
J
Y
HN
GE
S
W
F
T
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A
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V
P
L
M
I
51
V
N
C
J
L
A
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P
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H
K
W
M
D
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52 5354555657
X
IM
J
L
T
V
FY
D
C
W
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P
E
H
K
A
G
S
N
Q
58
H
59
AG
F
P
X
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H
T
K
E
M
W
V
N
I
J
L
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60
HW
6162
I
L
GK
R
63
R
G
ED
P
I
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L
T
V
H
F
N
A
C
64
S
A
GI
Y
T
F
LR
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P
V
J
W
K
N
C
X
H
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65
L
R
V
C
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QK
66
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67
I
D
SM
G
V
F
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L
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68
M
H
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69
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70
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71
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L
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G
H
N
72
L
R
E
NP
73
FE
R
Q
S
N
D
W
0
1
2
3
4
bits
-1
1
L
V
R
NX
P
2
V
Q
J
W
KR
E
D
L
H
F
N
G
T
3
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4
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5
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MW
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7
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89
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10
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11
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12
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13
L
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14Y
15
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16
C
I
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F
H
D
Y
S
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W
L
T
K
M
Q
V
P
N
A
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17
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VN
AL
M
T
RD
KP
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17a
SPX
181920
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P
E
H
K
L
T
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C
M
GS
Y
20a
L
R
P
I
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WE
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20b
LE
2122Y
23
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GP
C
L
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24
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25
D
YR
S
G
L
I
P
V
A
C
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T
W
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F
K
J
M
Q
H
N
26
PLQ
GJ
27
F
Y
X
N
C
Q
K
W
M
G
R
E
P
T
L
V
S
A
H
I
D
28
I
J
M
V
N
H
L
TG
F
C
S
A
R
P
Y
K
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E
D
X
W
29
G
EKL
P
M
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V
F
T
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N
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I
30
D
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MI
A
L
P
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31
FQP
L
V
T
S
R
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A
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D
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32
V
A
TH
M
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L
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3334
A
S
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L
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35
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W
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36
D
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3738
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39
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40
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T
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41
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T
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M
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D
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42
SI
P
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A
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L
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43
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T
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A
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E
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M
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Y
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44
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H
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45
P
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46
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Y
L
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47
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48
L
JE
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Y
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W
C
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A
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49
L
X
P
W
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T
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50
M
SE
F
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L
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W
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D
51
J
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G
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L
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52
L
M
TG
I
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535455
T
56575859
K
V
X
N
I
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A
LH
E
60
SR
M
G
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L
FN
T
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H
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616263
I
N
F
XC
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A
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L
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T
M
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D
64
V
K
XS
C
W
G
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T
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M
P
H
L
A
F
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65
E
CG
Q
V
A
K
R
H
L
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P
Y
T
X
F
J
W
M
I
N
66
Y
T
W
V
AK
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F
I
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G
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R
L
M
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N
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D
67
G
A
J
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T
K
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L
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I
M
Y
N
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X
F
68
R
A
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H
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N
L
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M
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P
Y
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X
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69
R
V
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P
N
D
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Y
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S
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70
M
SRH
Y
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L
J
V
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D
K
W
71
T
G
N
L
F
D
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K
M
W
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X
V
P
S
Y
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72
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HM
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L
I
V
K
S
F
R
C
A
Y
D
T
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73
A
P
N
HX0
1
2
3
4
bits
-1
1
L
IXN
Q
2
LK
H
E
R
3
V
L
XM
SR
E
T
4
X
G
L
P
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T
SR
F
M
N
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5
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M
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6
Y
MR
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D
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7
G
PF
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M
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A
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8
Y
C
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10
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S
11
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12
L
Y
S
PQ
G
T
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A
W
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13
T
X
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PG
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14 15
T
VE
16
E
XR
A
N
P
V
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T
Y
K
F
L
W
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C
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17
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G
PN
V
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T
M
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F
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L
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A
I
17a
G
IF
W
XP
18 19 20
AV
Y
S
GM
C
F
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T
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L
H
E
P
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D
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N
20a
L
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W
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21
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22
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23
LR
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24X
25
A
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26
L
M
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27
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M
D
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28
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29
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A
30
L
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31
AM
GR
L
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P
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32
Y
X
WM
SR
C
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A
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33 34
S
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35
D
Y
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36
S
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37 38
G
HR
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39
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A
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H
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40
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41
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42
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A
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43
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44
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45
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46
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47
L
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A
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48
A
HC
G
W
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49
LR
H
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50
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C
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A
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51
R
K
T
L
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H
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52
G
JR
P
HT
53 54 55 56
A
57 58 59
P
J
WH
M
Q
R
L
K
Y
N
T
V
G
F
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A
C
60
D
A
H
V
W
KT
N
F
L
Q
G
M
R
S
C
Y
X
E
P
I
J
61
GH
R
62
XF
S
E
K
J
H
W
R
M
G
C
A
P
L
I
V
T
63
S
G
AT
Y
J
L
Q
K
V
N
W
P
R
I
X
64
C
QI
N
Y
F
P
L
R
A
K
W
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V
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65
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Y
IW
H
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L
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A
G
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D
66
CHV
JR
N
M
P
L
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A
S
I
F
E
T
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Y
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67
R
AN
C
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W
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L
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D
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68
R
G
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A
D
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H
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69
V
L
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CM
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70
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FMVH
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71
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DR
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72
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XSK
V
I
LJ
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M
H
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73
L
I
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QG
C
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A
Rhizobiales + Rhodobacteriaceae + Caulobacterales +
Hyphomonadaceae (RRCH)
Rickettsiales +Sphingomonadales + Rhodospirialles (RSR)
U
C
G
A
genome
functionally classified tRNAs
X R C E
J E Y
Score each tRNA
sequence for taxon-specific
functional Class-Informative-
Features (CIFs)
Average scores across tRNAs.
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM 0
1
2
3
4
bits
-1
1
C
I
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2E
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3
VKR
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Y
4
I
M
L
R
D
Y
J
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5
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N
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6
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7
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89
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10
SG
Y
L
11X
12
PT
G
SC
13
L
C
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1415
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16
T
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C
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17
L
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SR
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1819R
20
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AR
20aL
20b
RY
21
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22
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23
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V
A
W
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H
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24
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25S
26
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T
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N
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27
A
G
C
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X
Y
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L
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M
K
F
Q
N
28
A
M
V
J
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N
L
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F
I
29
K
TA
D
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M
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H
Y
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G
30
S
T
31
VH
SP
N
Q
A
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L
T
M
K
C
Y
F
32
AM
3334R
35
X
V
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36
L
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F
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37
LQ
R
V
S
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Y
A
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DI
F
G
J
M
N
K
C
38
D
HL
AP
T
G
S
R
Y
W
X
F
K
J
M
C
I
N
39
VI
A
R
T
LP
H
Q
S
W
40
T
41
L
S
HF
R
J
D
N
P
T
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A
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42
H
I
D
S
M
W
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G
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A
L
N
R
K
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V
43
LQJ
N
S
T
E
G
K
F
P
D
W
A
V
R
H
X
44
L
SM
C
N
K
P
J
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H
W
R
A
I
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D
X
45
R
A
W
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M
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L
J
V
S
G
C
Q
E
46
S
T
W
Y
Q
L
G
C
E
47
L
Q
DIY
48
G
A
V
R
TD
49
IL
G
F
P
Y
J
S
K
H
X
T
W
C
R
V
A
E
50
RL
I
W
V
X
T
Q
K
A
Y
S
G
51
AT
W
N
K
V
L
P
R
X
Y
J
Q
I
52
S
QG
A
W
K
Y
RD
P
V
H
L
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I
5354555657
SG
AK
H
E
P
R
W
C
D
Y
F
V
T
L
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M
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5859
CL
F
W
A
I
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V
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N
K
M
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ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
ACC STEM T STEMT STEM T LOOPAC STEM AC STEMACD STEM D STEMD LOOP ACC STEM
tRNA Signatures Reveal a Polyphyletic Origin of SAR11
Strains among Alphaproteobacteria
Katherine C. H. Amrine, Wesley D. Swingley
€
, David H. Ardell*
Program in Quantitative and Systems Biology, University of California, Merced, Merced, California, United States of America
Abstract
Molecular phylogenetics and phylogenomics are subject to noise from horizontal gene transfer (HGT) and bias from
convergence in macromolecular compositions. Extensive variation in size, structure and base composition of
alphaproteobacterial genomes has complicated their phylogenomics, sparking controversy over the origins and closest
relatives of the SAR11 strains. SAR11 are highly abundant, cosmopolitan aquatic Alphaproteobacteria with streamlined, A+T-
biased genomes. A dominant view holds that SAR11 are monophyletic and related to both Rickettsiales and the ancestor of
mitochondria. Other studies dispute this, finding evidence of a polyphyletic origin of SAR11 with most strains distantly
related to Rickettsiales. Although careful evolutionary modeling can reduce bias and noise in phylogenomic inference,
entirely different approaches may be useful to extract robust phylogenetic signals from genomes. Here we develop simple
phyloclassifiers from bioinformatically derived tRNA Class-Informative Features (CIFs), features predicted to target tRNAs for
specific interactions within the tRNA interaction network. Our tRNA CIF-based model robustly and accurately classifies
alphaproteobacterial genomes into one of seven undisputed monophyletic orders or families, despite great variability in
tRNA gene complement sizes and base compositions. Our model robustly rejects monophyly of SAR11, classifying all but
one strain as Rhizobiales with strong statistical support. Yet remarkably, conventional phylogenetic analysis of tRNAs
classifies all SAR11 strains identically as Rickettsiales. We attribute this discrepancy to convergence of SAR11 and
Rickettsiales tRNA base compositions. Thus, tRNA CIFs appear more robust to compositional convergence than tRNA
sequences generally. Our results suggest that tRNA-CIF-based phyloclassification is robust to HGT of components of the
tRNA interaction network, such as aminoacyl-tRNA synthetases. We explain why tRNAs are especially advantageous for
prediction of traits governing macromolecular interactions from genomic data, and why such traits may be advantageous in
the search for robust signals to address difficult problems in classification and phylogeny.
Citation:Amrine KCH, Swingley WD, Ardell DH (2014) tRNA Signatures Reveal a Polyphyletic Origin of SAR11 Strains among Alphaproteobacteria. PLoS Comput
Biol 10(2): e1003454. doi:10.1371/journal.pcbi.1003454
Editor:Christos A. Ouzounis, The Centre for Research and Technology, Hellas, Greece
ReceivedMay 1, 2013;AcceptedDecember 10, 2013;PublishedFebruary 27, 2014
Copyright:!2014 Amrine et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:This work was supported by UC Merced. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests:The authors have declared that no competing interests exist.
* E-mail: [email protected]
€ Current address: Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, United States of America.
Introduction
Which parts of genomes are most resistant to compositional
convergence? Which information is vertically inherited most
faithfully? Compositional stationarity and vertical (co-)inheritance
are key, yet frequently violated, assumptions of most current
approaches in molecular phylogenetics and phylogenomics [1].
Horizontal gene transfer (HGT), for example, is so common and
widespread that the very existence of a ‘‘Tree of Life’’ has been
called into question [2,3]. Advances in understanding the history
of life will require discovery of new universal, slowly-evolving
phylogenetic markers that are resistant to compositional conver-
gence and HGT.
The controversial phylogeny ofCa.Pelagibacter ubique
(SAR11) is a case in point. SAR11 make up between a fifth and
a third of the bacterial biomass in marine and freshwater
ecosystems [4]. SAR11 have very small cell sizes, genome sizes,
and intergenic region sizes, possibly in adaptation to extreme
nutrient limitations [5]. Some recent phylogenomic studies place
free-living SAR11 together in a clade with the largely endopar-
asitic Rickettsiales and the alphaproteobacterial ancestor of
mitochondria [6,7,8]. Other studies persuasively argue that this
placement is an artifact of independent convergence of SAR11
and Rickettsiales towards increased genomic A+T contents, and
that SAR11 are more closely related to the free-living Alphaproteo-
bacteria such as the Rhizobiales and Rhodobacteraceae [9,10,11].
The monophyly of SAR11 was also recently rejected [10,12].
Nonstationary macromolecular compositions are a known
source of bias in phylogenomics [13,14]. Widespread variation
in macromolecular compositions may be caused by loss of DNA
repair pathways in reduced genomes [15,11], unveiling an
inherent A+T-bias of mutation in bacteria [16] that elevates
genomic A+T contents [17,18]. A process such as this has likely
altered protein and RNA compositions genome-wide in SAR11,
and if such effects are accounted for, SAR11 appear more closely
related to Rhizobiales and Rhodobacteraceae than Rickettsiales
[10,11]. Consistent with this interpretation, SAR11 strain
HTTC1062 shares, with a large clade of free-living Alphaproteo-
bacteria that excludes the Rickettsiales, a unique and derived
codivergence of features that govern recognition between
tRNAHis and histidyl-tRNA synthetase (HisRS) [19,20]. This
unique functionally significant synapomorphy likely arose only
PLOS Computational Biology | www.ploscompbiol.org 1 February 2014 | Volume 10 | Issue 2 | e1003454
Katie Amrine, Ph.D.
tRNA Signatures Reveal a Polyphyletic Origin of SAR11
Strains among Alphaproteobacteria
Katherine C. H. Amrine, Wesley D. Swingley
€
, David H. Ardell*
Program in Quantitative and Systems Biology, University of California, Merced, Merced, California, United States of America
Abstract
Molecular phylogenetics and phylogenomics are subject to noise from horizontal gene transfer (HGT) and bias from
convergence in macromolecular compositions. Extensive variation in size, structure and base composition of
alphaproteobacterial genomes has complicated their phylogenomics, sparking controversy over the origins and closest
relatives of the SAR11 strains. SAR11 are highly abundant, cosmopolitan aquatic Alphaproteobacteria with streamlined, A+T-
biased genomes. A dominant view holds that SAR11 are monophyletic and related to both Rickettsiales and the ancestor of
mitochondria. Other studies dispute this, finding evidence of a polyphyletic origin of SAR11 with most strains distantly
related to Rickettsiales. Although careful evolutionary modeling can reduce bias and noise in phylogenomic inference,
entirely different approaches may be useful to extract robust phylogenetic signals from genomes. Here we develop simple
phyloclassifiers from bioinformatically derived tRNA Class-Informative Features (CIFs), features predicted to target tRNAs for
specific interactions within the tRNA interaction network. Our tRNA CIF-based model robustly and accurately classifies
alphaproteobacterial genomes into one of seven undisputed monophyletic orders or families, despite great variability in
tRNA gene complement sizes and base compositions. Our model robustly rejects monophyly of SAR11, classifying all but
one strain as Rhizobiales with strong statistical support. Yet remarkably, conventional phylogenetic analysis of tRNAs
classifies all SAR11 strains identically as Rickettsiales. We attribute this discrepancy to convergence of SAR11 and
Rickettsiales tRNA base compositions. Thus, tRNA CIFs appear more robust to compositional convergence than tRNA
sequences generally. Our results suggest that tRNA-CIF-based phyloclassification is robust to HGT of components of the
tRNA interaction network, such as aminoacyl-tRNA synthetases. We explain why tRNAs are especially advantageous for
prediction of traits governing macromolecular interactions from genomic data, and why such traits may be advantageous in
the search for robust signals to address difficult problems in classification and phylogeny.
Citation:Amrine KCH, Swingley WD, Ardell DH (2014) tRNA Signatures Reveal a Polyphyletic Origin of SAR11 Strains among Alphaproteobacteria. PLoS Comput
Biol 10(2): e1003454. doi:10.1371/journal.pcbi.1003454
Editor:Christos A. Ouzounis, The Centre for Research and Technology, Hellas, Greece
ReceivedMay 1, 2013;AcceptedDecember 10, 2013;PublishedFebruary 27, 2014
Copyright:!2014 Amrine et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:This work was supported by UC Merced. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests:The authors have declared that no competing interests exist.
* E-mail: [email protected]
€ Current address: Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, United States of America.
Introduction
Which parts of genomes are most resistant to compositional
convergence? Which information is vertically inherited most
faithfully? Compositional stationarity and vertical (co-)inheritance
are key, yet frequently violated, assumptions of most current
approaches in molecular phylogenetics and phylogenomics [1].
Horizontal gene transfer (HGT), for example, is so common and
widespread that the very existence of a ‘‘Tree of Life’’ has been
called into question [2,3]. Advances in understanding the history
of life will require discovery of new universal, slowly-evolving
phylogenetic markers that are resistant to compositional conver-
gence and HGT.
The controversial phylogeny ofCa.Pelagibacter ubique
(SAR11) is a case in point. SAR11 make up between a fifth and
a third of the bacterial biomass in marine and freshwater
ecosystems [4]. SAR11 have very small cell sizes, genome sizes,
and intergenic region sizes, possibly in adaptation to extreme
nutrient limitations [5]. Some recent phylogenomic studies place
free-living SAR11 together in a clade with the largely endopar-
asitic Rickettsiales and the alphaproteobacterial ancestor of
mitochondria [6,7,8]. Other studies persuasively argue that this
placement is an artifact of independent convergence of SAR11
and Rickettsiales towards increased genomic A+T contents, and
that SAR11 are more closely related to the free-living Alphaproteo-
bacteria such as the Rhizobiales and Rhodobacteraceae [9,10,11].
The monophyly of SAR11 was also recently rejected [10,12].
Nonstationary macromolecular compositions are a known
source of bias in phylogenomics [13,14]. Widespread variation
in macromolecular compositions may be caused by loss of DNA
repair pathways in reduced genomes [15,11], unveiling an
inherent A+T-bias of mutation in bacteria [16] that elevates
genomic A+T contents [17,18]. A process such as this has likely
altered protein and RNA compositions genome-wide in SAR11,
and if such effects are accounted for, SAR11 appear more closely
related to Rhizobiales and Rhodobacteraceae than Rickettsiales
[10,11]. Consistent with this interpretation, SAR11 strain
HTTC1062 shares, with a large clade of free-living Alphaproteo-
bacteria that excludes the Rickettsiales, a unique and derived
codivergence of features that govern recognition between
tRNAHis and histidyl-tRNA synthetase (HisRS) [19,20]. This
unique functionally significant synapomorphy likely arose only
PLOS Computational Biology | www.ploscompbiol.org 1 February 2014 | Volume 10 | Issue 2 | e1003454
ACC STEM
U73 conveys
about six times
more information
about tRNA
function than A73
U73 is equally over-
represented in tRNA
Cys
and tRNA
Gly
8597 tRNA genes from 147 alphaproteobacterial genomes
The height of a letter
quantifies both
functional information
of a CIF and its
relative over-
representation in a
particular functional
class of tRNAs.
Function logos visualize information about
function, conditioned by a common structure
Visualizing bacterial tRNA identity determinants and
antideterminants using function logos and inverse
function logos
Eva Freyhult, Vincent Moulton
1
and David H. Ardell*
Linnaeus Centre for Bioinformatics, Uppsala University, Uppsala, Sweden and
1
School of Computing Sciences,
University of East Anglia, Norwich NR4 7TJ, UK
Received November 21, 2005; Revised and Accepted January 12, 2006
ABSTRACT
Sequence logos are stacked bar graphs that gen-
eralize the notion of consensus sequence. They
employ entropy statistics very effectively to display
variation in a structural alignment of sequences
of a common function, while emphasizing its over-
represented features. Yet sequence logos cannot
display features that distinguish functional sub-
classes within a structurally related superfamily
nor do they display under-represented features. We
introduce two extensions to address these needs:
function logos and inverse logos. Function logos
display subfunctions that are over-represented
among sequences carrying a specific feature.
Inverse logos generalize both sequence logos and
function logos by displaying under-represented,
rather than over-represented, features or functions
in structural alignments. To make inverse logos,
a compositional inverse is applied to the feature
or function frequency distributions before logo
construction, where a compositional inverse is a
mathematical transform that makes common fea-
tures or functions rare andvice versa. We applied
these methods to a database of structurally aligned
bacterial tDNAs to create highly condensed, birds-
eye views of potentially all so-called identity
determinants and antideterminants that confer spe-
cific amino acid charging or initiator function on
tRNAs in bacteria. We recovered both known
and a few potentially novel identity elements.
Function logos and inverse logos are useful tools
for exploratory bioinformatic analysis of structure–
function relationships in sequence families and
superfamilies.
INTRODUCTION
Which sequence features confer a specific biological function
on a class of macromolecules? This question becomes both
more acute and more tractable when contrasting classes of
molecules with distinct functions yet highly similar structures.
In that situation, there may be fewer structural differences
to explain the functional differences among classes, but this
also means fewer structural differences to detect and test.
Highly similar structures also make structural analogy easier
to assign. A common complication arises, however, in that
structural similarity may derive from common ancestry rather
than from functional constraint.
An example of such a problem is transfer RNA (tRNA)
identity [reviewed in (1–4)]. A pillar of fidelity in gene exp-
ression is the consistency with which specific amino acids are
attached to specific tRNAs by enzymes called aminoacyl-
tRNA synthetases (aaRSs). In general, there is one population
of aaRS in a cell for each of the 20 canonical amino acids.
Despite the generally very high structural similarity of all
tRNAs, each must interact productively with only one syn-
thetase population to be charged with its cognate amino acid
and interact nonproductively with the remaining 19 enzyme
populations. Theidentityof a tRNA refers to this amino acid
‘charging’ specificity. In a simplification, the identity of a
tRNA can be thought to depend on a constellation of structural
features called ‘identity elements,’ encompassing features that
promote recognition and catalytic activity by its cognate aaRS
(called ‘determinants’) or discrimination by noncognate
synthetases against the same or other features (called ‘anti-
determinants’) so as to inhibit translationally ambiguous
tRNA-binding and aminoacylation.
We (roughly) define a ‘tRNA identity code’ as the set of all
identity elements that make tRNAs functionally distinct within
a taxonomic lineage. We note five points about identity codes
as described in more detail in the aforementioned reviews.
First, a complete identity code has never been completely
described for any taxonomic lineage. Second, identity codes
*To whom correspondence should be addressed at David Ardell, Linnaeus Centre for Bioinformatics, Box 598, 751 24 Uppsala, Sweden. Tel: +46 18 471 6694;
Fax: +46 18 471 6698; E-mail: [email protected]
!The Author 2006. Published by Oxford University Press. All rights reserved.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access
version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and OxfordUniversity Press
are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but
only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected]
Nucleic Acids Research, 2006, Vol. 34, No. 3905–916
doi:10.1093/nar/gkj478
Published online February 9, 2006
Eva Freyhult, Ph.D.
Instead of a Conservation Criterion to Predict
Functionality, CIFs use an Information Criterion
Uncertainty of
features over all tRNAs
Uncertainty of
features over tRNAs
with given function Y
H(X)−H(X|Y)
Conservation Criterion (sequence logos)
“Given that we know the function Y of a tRNA, how much
information do we gain about its features?”
“Given that a protein recognizes feature X in a tRNA, how much
information does it gain about its functions?”
Uncertainty of
functions over all tRNAs
Uncertainty of
functions over tRNAs
with given feature X
H(Y)−H(Y|X)
Information Criterion (function logos)
Functionality, CIFs use an Information Criterion
Uncertainty of
features over all tRNAs
Uncertainty of
features over tRNAs
with given function Y
H(X)−H(X|Y)
Conservation Criterion (sequence logos)
“Given that we know the function Y of a tRNA, how much
information do we gain about its features?”
“Given that a protein recognizes feature X in a tRNA, how much
information does it gain about its functions?”
Uncertainty of
functions over all tRNAs
Uncertainty of
functions over tRNAs
with given feature X
H(Y)−H(Y|X)
Information Criterion (function logos)
Function Logos of Bacterial tRNAs from tRNAdb
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Adenine
Cytosine
Guanine
Uracil
gap
Freyhult et al. (2006) Nucleic Acids Research 34(3):915
Asn-tRNAs
Phe-tRNAs
His-tRNAs
Leu-tRNAs
Trp-tRNAs
Tyr-tRNAs
ATCG
N
N
N
N
N
N
F
F
F
F
F
F
H
H
H
H
H
H
L
L
L
L
L
L
W
W
W
W
W
Y
Y
Y
Y
Y
Y
To compute the significance of tRNA CIFs,
permute class labels over sequences
Phe-tRNAs
His-tRNAs
Leu-tRNAs
Trp-tRNAs
Tyr-tRNAs
ATCG
N
N
N
N
N
N
F
F
F
F
F
F
H
H
H
H
H
H
L
L
L
L
L
L
W
W
W
W
W
Y
Y
Y
Y
Y
Y
To compute the significance of tRNA CIFs,
permute class labels over sequences
To compute the significance of tRNA CIFs,
permute class labels over sequences
Asn-tRNAs
Phe-tRNAs
His-tRNAs
Leu-tRNAs
Trp-tRNAs
Tyr-tRNAs
ATCG
N
N
N
N
N
N
F
F
F
F
F
F
H
H
H
H
H
H
L
L
L
L
L
L
W
W
W
W
W
Y
Y
Y
Y
Y
Y
permute class labels over sequences
Asn-tRNAs
Phe-tRNAs
His-tRNAs
Leu-tRNAs
Trp-tRNAs
Tyr-tRNAs
ATCG
N
N
N
N
N
N
F
F
F
F
F
F
H
H
H
H
H
H
L
L
L
L
L
L
W
W
W
W
W
Y
Y
Y
Y
Y
Y
0
1
2
3
4
bits
0
K
Y
123
YQWMVPRHKXSEGINLFCTA
456
KQ
DWHFNYCRPGVI
ASLTEXM
789
10 11 12 13 14 15 16 17 18 19 20 21 22
R
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23
LPXKFGNWMCIRDYSET
A
24 25
SARI
E
T
W
H
P
L
26 2728 29 30
S
WHT
C
EYGLPFM
DNRIQVKAX
31 32 33 34 35
C
KQWHDAEP
INMGLFRSYT
VX
36 37 38
IATXPK
Y
R
S
W
39 40
LGIPEAM
C
T
D
41 42 43 44 45
FDYWSAGHXMVCR
KI
ENLPT
46
ARGLV
47
X
E
F
QK
I
T
DY
PN
R
S
VAHG
L
CWM
48
F
HKLYVGQIWEPR
T
D
XANSC
49
W
H
XCD
SEMRQLNIAPFVTYGK
50 51 52 53 54 55
A
G
IS
KT
LQ
RXFN
C
56 57 58 59 60 61 62 63 64 65
DQL
IMCEFARPVGKTXNS
66 67
ISALN
68 69
LRX
S
K
A
P
70
LSGREFIX
71 72
I
G
AK
YQRLPVFTSE
XN
73 74
GSNYQW
MEDAITLKCXFRV
75
LGP
K
76 77
E
CIKNXHPFGS
TAWDYVLRQM
78
Q
HDYRXLEFTIG
KSNVAMP
79 80 81 82 83 84 85 86 87
FQWDYVR
HTGPMXAESKNLIC
88 89 90 91 92 93
E
F
H
G
X
94 95 9697
EGYCF
TARQXSLNPVIWKH
98 99
ALSV
TDQIMCREXY
100 101 102 103 104
YQMD
SWNHXCATPKEVLRGIF
105
0
1
2
3
4
bits
01
A
R
L
P
G
I
MCTSDX
WNYQ
2
A
S
VTDLIMCREXYQ
3456789
10 11
S
W
T
HYEPGF
LMRDNIQVAKX
12 13 14
P
SHTMACLGKNYQVRXDI
15 16 17
AV
D
X
18
MNH
DVIQSAG
LEWCKXRFYP
T
19 20 21 22 23 24
C
M
NYXSFPDHVGIWEKQA
RLT
25 26 27
SRPMF
28 29 30 31
G
PVRKFNDL
MSCT
IQXHYA
32 33
H
C
QWES
XDFNLRIMATYGVKP
34
MF
HK
L
YGI
WE
RVQPDXATNSC
35
X
E
F
QK
I
T
D
P
Y
SN
RVAG
L
C
W
H
M
36
RGTXANLQV
37 38 39 40
WFQ
YXTPEKHDCISRLAMNVG
41 42 43 44 45 46
SR
AFGDYIXP
CN
47
C
Q
W
KD
AEPINH
SMGLFYR
TVX
48 49 50 51 52 53 54 55
LRGAKIYSX
F
56
G
I
AK
YQRPVCLFTS
E
NX
57
TIXSAV
KYRLGPC
58 59
IP
A
F
L
Y
60
ARLXSEG
C
61
LRI
A
N
X
62
PKGDIHMVRQSEAFTLX
63
K
F
DNWMITRSVALEGXP
64
IARSVE
F
T
N
L
65 66
S
L
67 68 69 70
LI
T
X
VRGNKP
71 72
A
IKNQGXMPCY
73
G
IPKDLYQARSNTFXC
74
IXAKTDQ
RHGEYWSVNLP
75 76 77
SAE
F
H
G
X
78
D
PRHKCIFVXSL
TMEANYQWG
79 80 81 82 83 84 85 86 87 88 89 90 91 92
HCQXFR
YTEIDLVNPGKWMAS
93
C
N
P
W
K
E
I
TS
G
XA
F
QYR
HL
DMV
94 95
Q
KD
WHFNYRGPVIACSL
TEMX
9697 98
DYHPQ
EXKMWFRVNITLCSGA
99
GTISDNY
ARMPVLF
100 101 102 103 104 105
0
1
2
3
4
bits
012
CXMVRFTQYEDGWAI
K
N
P
S
L
H
345
K
F
X
W
P
MCD
H
IV
T
R
A
L
SGYQNE
6789
10 11
SA
V
C
12 1314 15 16 17 18
S
G
EFTLYPIRKDWXH
19 20 21 22 23
T
E
KDNYQIHALWSMRCGV
24
S
A
N
25
PELISCRGKFNXAT
26 2728 29 30 31
XSVFKGNIWRHACELTP
32 33 34 35 36 37 38 39 40 41
Y
EPLXFQWCV
I
HAKT
N
R
DSGM
42 43
H
C
EF
RI
GKWALDQXPSNYTV
44 45
FM
PTNWDYKELSQGVICXHAR
46
PGNDWAK
Y
HMELF
VCS
ITQRX
47 48 49 50
D
Q
R
K
A
HT
MX
E
NSFIYVG
LPCW
51
X
N
F
YQ
V
L
R
G
W
K
PE
AITCDMSH
52 53 54 55
F
HS
C
I
KDYQRLMNVGXATE
P
56
XPVELHTM
SCIKDANRG
57
L
I
S
VN
D
RGMC
AF
TPKX
58
ISFGDNYTCAREKX
59
L
IPFTYQ
GSAEKNRX
60 61 62 63 64
R
P
G
65 66 67
R
G
I
X
68
L
SAR
EG
X
69
G
L
I
A
70
R
XFD
ISGTMVCLNYQPA
71
DYHMGS
NQVRIEFTAXPL
72
VNIQSPCREAXL
T
F
73
IAG
T
XRLNE
74 75
AGTXN
DSP
76 77
Q
M
VR
L
Y
WHPFT
DAG
S
X
N
K
E
I
C
78 79
YCHGPWRM
SETIKQNLFAXDV
80 81 82 83 84 85 86 87 88 89 90 91 92 9394 95 9697 98 99
100
A
PXVRK
T
L
SHGDNI
YC
101
DWRELPF
GHYQSXMITKANC
102 103 104 105
0
1
2
3
4
bits
01234
NGHVWP
KCIQSRFMTEXLDAY
5
E
N
K
H
XD
A
Y
G
R
ST
W
IVMQ
L
P
CF
67
ST
XPA
FYRVQ
89
10
XSVFKGNIWRHACELTP
11 12 1314 15 16 17 18 19
XPRGD
LEKSQVHTNI
AWFY
20 21 22 23
C
KSDYWAF
NXVQREGIHPLT
M
24 25 26 27 28
L
W
29 30
SA
V
C
31 32 33 34 35 36
P
NGDWAKVYHLQMEFCTSI
R
X
37
FWP
TN
D
YMK
Q
ELSGICVXHAR
38
D
W
QY
S
XV
K
F
M
T
RG
L
N
P
I
E
AHC
39 40 41
Q
WSHKPDYLIVGMR
NETXCFA
42
P
NDQWEFHTCYX
KIMAGSRLV
43 44
TQ
HPCYIFXNMARKVDGSL
45 46 47 48 49 50 51
I
A
G
L
52 53 54 55
SRTPXEL
56
SGIVQRXAE
L
T
F
P
57
GDYHMSV
NIRQPAFTLXE
58
RTXVGDISCLNQP
A
59
I
S
G
A
P
60 61 62
S
R
63 64
ILSPXEFDWA
65 66
AT
S
V
67 68
IRXEFTAC
69 70
I
SFKGDNQRTCAEX
71 72
XSVELIHTMPCRKADG
73
D
HSCI
KNYQRLGATXMVEP
74
G
T
X
P
VRIQWK
75
LXAE
F
N
S
I
T
C
76
XFMRVATPYNDCGEQKL
W
HSI
77 78 79 80 81 82 83 84 85 86 87 88 89 90 91
YCPREGWHMSFNTQ
I
KADL
XV
92 93
VHQMLYFDR
XTAEGPKWSINC
94 95
V
T
X
C
H
YR
I
W
S
GA
ELN
Q
P
D
K
F
9697 98 99
CXFMRVYTDEGQAIWNSKLPH
100
DCNYKWQISX
PM
GHTFLAE
V
R
101
LIKQVD
Y
W
E
102
YDQSMWNHCAEXPTKVLRG
I
F
103
YDQSMWNHCAEXPTKVLRG
I
F
104 105
0
1
2
3
4
bits
01
L
X
Y
23456789
10 11 12 13 14 15 16 1718 19
M
W
GYQHSE
CKTDV
PI
ALF
RNX
20 21 22 23 24 25 26 2728 29 30 31 32 33 34 35 36 37 38 39 40 41 42 4344 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
LIRGFTQXSAEN
75
H
S
PL
KCDGYTEQMRI
NAVFX
76 77 78 79 80 81 82 83 8485 8687
S
G
L
8889 9091 92 9394 95 9697 98 99
100 101
TL
V
Q
X
102 103 104 105
Logos from the same data randomized through permutation
Features with 0.5 bits are significant at the 1% level
1
2
3
4
bits
0
K
Y
123
YQWMVPRHKXSEGINLFCTA
456
KQ
DWHFNYCRPGVI
ASLTEXM
789
10 11 12 13 14 15 16 17 18 19 20 21 22
R
G
S
23
LPXKFGNWMCIRDYSET
A
24 25
SARI
E
T
W
H
P
L
26 2728 29 30
S
WHT
C
EYGLPFM
DNRIQVKAX
31 32 33 34 35
C
KQWHDAEP
INMGLFRSYT
VX
36 37 38
IATXPK
Y
R
S
W
39 40
LGIPEAM
C
T
D
41 42 43 44 45
FDYWSAGHXMVCR
KI
ENLPT
46
ARGLV
47
X
E
F
QK
I
T
DY
PN
R
S
VAHG
L
CWM
48
F
HKLYVGQIWEPR
T
D
XANSC
49
W
H
XCD
SEMRQLNIAPFVTYGK
50 51 52 53 54 55
A
G
IS
KT
LQ
RXFN
C
56 57 58 59 60 61 62 63 64 65
DQL
IMCEFARPVGKTXNS
66 67
ISALN
68 69
LRX
S
K
A
P
70
LSGREFIX
71 72
I
G
AK
YQRLPVFTSE
XN
73 74
GSNYQW
MEDAITLKCXFRV
75
LGP
K
76 77
E
CIKNXHPFGS
TAWDYVLRQM
78
Q
HDYRXLEFTIG
KSNVAMP
79 80 81 82 83 84 85 86 87
FQWDYVR
HTGPMXAESKNLIC
88 89 90 91 92 93
E
F
H
G
X
94 95 9697
EGYCF
TARQXSLNPVIWKH
98 99
ALSV
TDQIMCREXY
100 101 102 103 104
YQMD
SWNHXCATPKEVLRGIF
105
0
1
2
3
4
bits
01
A
R
L
P
G
I
MCTSDX
WNYQ
2
A
S
VTDLIMCREXYQ
3456789
10 11
S
W
T
HYEPGF
LMRDNIQVAKX
12 13 14
P
SHTMACLGKNYQVRXDI
15 16 17
AV
D
X
18
MNH
DVIQSAG
LEWCKXRFYP
T
19 20 21 22 23 24
C
M
NYXSFPDHVGIWEKQA
RLT
25 26 27
SRPMF
28 29 30 31
G
PVRKFNDL
MSCT
IQXHYA
32 33
H
C
QWES
XDFNLRIMATYGVKP
34
MF
HK
L
YGI
WE
RVQPDXATNSC
35
X
E
F
QK
I
T
D
P
Y
SN
RVAG
L
C
W
H
M
36
RGTXANLQV
37 38 39 40
WFQ
YXTPEKHDCISRLAMNVG
41 42 43 44 45 46
SR
AFGDYIXP
CN
47
C
Q
W
KD
AEPINH
SMGLFYR
TVX
48 49 50 51 52 53 54 55
LRGAKIYSX
F
56
G
I
AK
YQRPVCLFTS
E
NX
57
TIXSAV
KYRLGPC
58 59
IP
A
F
L
Y
60
ARLXSEG
C
61
LRI
A
N
X
62
PKGDIHMVRQSEAFTLX
63
K
F
DNWMITRSVALEGXP
64
IARSVE
F
T
N
L
65 66
S
L
67 68 69 70
LI
T
X
VRGNKP
71 72
A
IKNQGXMPCY
73
G
IPKDLYQARSNTFXC
74
IXAKTDQ
RHGEYWSVNLP
75 76 77
SAE
F
H
G
X
78
D
PRHKCIFVXSL
TMEANYQWG
79 80 81 82 83 84 85 86 87 88 89 90 91 92
HCQXFR
YTEIDLVNPGKWMAS
93
C
N
P
W
K
E
I
TS
G
XA
F
QYR
HL
DMV
94 95
Q
KD
WHFNYRGPVIACSL
TEMX
9697 98
DYHPQ
EXKMWFRVNITLCSGA
99
GTISDNY
ARMPVLF
100 101 102 103 104 105
0
1
2
3
4
bits
012
CXMVRFTQYEDGWAI
K
N
P
S
L
H
345
K
F
X
W
P
MCD
H
IV
T
R
A
L
SGYQNE
6789
10 11
SA
V
C
12 1314 15 16 17 18
S
G
EFTLYPIRKDWXH
19 20 21 22 23
T
E
KDNYQIHALWSMRCGV
24
S
A
N
25
PELISCRGKFNXAT
26 2728 29 30 31
XSVFKGNIWRHACELTP
32 33 34 35 36 37 38 39 40 41
Y
EPLXFQWCV
I
HAKT
N
R
DSGM
42 43
H
C
EF
RI
GKWALDQXPSNYTV
44 45
FM
PTNWDYKELSQGVICXHAR
46
PGNDWAK
Y
HMELF
VCS
ITQRX
47 48 49 50
D
Q
R
K
A
HT
MX
E
NSFIYVG
LPCW
51
X
N
F
YQ
V
L
R
G
W
K
PE
AITCDMSH
52 53 54 55
F
HS
C
I
KDYQRLMNVGXATE
P
56
XPVELHTM
SCIKDANRG
57
L
I
S
VN
D
RGMC
AF
TPKX
58
ISFGDNYTCAREKX
59
L
IPFTYQ
GSAEKNRX
60 61 62 63 64
R
P
G
65 66 67
R
G
I
X
68
L
SAR
EG
X
69
G
L
I
A
70
R
XFD
ISGTMVCLNYQPA
71
DYHMGS
NQVRIEFTAXPL
72
VNIQSPCREAXL
T
F
73
IAG
T
XRLNE
74 75
AGTXN
DSP
76 77
Q
M
VR
L
Y
WHPFT
DAG
S
X
N
K
E
I
C
78 79
YCHGPWRM
SETIKQNLFAXDV
80 81 82 83 84 85 86 87 88 89 90 91 92 9394 95 9697 98 99
100
A
PXVRK
T
L
SHGDNI
YC
101
DWRELPF
GHYQSXMITKANC
102 103 104 105
0
1
2
3
4
bits
01234
NGHVWP
KCIQSRFMTEXLDAY
5
E
N
K
H
XD
A
Y
G
R
ST
W
IVMQ
L
P
CF
67
ST
XPA
FYRVQ
89
10
XSVFKGNIWRHACELTP
11 12 1314 15 16 17 18 19
XPRGD
LEKSQVHTNI
AWFY
20 21 22 23
C
KSDYWAF
NXVQREGIHPLT
M
24 25 26 27 28
L
W
29 30
SA
V
C
31 32 33 34 35 36
P
NGDWAKVYHLQMEFCTSI
R
X
37
FWP
TN
D
YMK
Q
ELSGICVXHAR
38
D
W
QY
S
XV
K
F
M
T
RG
L
N
P
I
E
AHC
39 40 41
Q
WSHKPDYLIVGMR
NETXCFA
42
P
NDQWEFHTCYX
KIMAGSRLV
43 44
TQ
HPCYIFXNMARKVDGSL
45 46 47 48 49 50 51
I
A
G
L
52 53 54 55
SRTPXEL
56
SGIVQRXAE
L
T
F
P
57
GDYHMSV
NIRQPAFTLXE
58
RTXVGDISCLNQP
A
59
I
S
G
A
P
60 61 62
S
R
63 64
ILSPXEFDWA
65 66
AT
S
V
67 68
IRXEFTAC
69 70
I
SFKGDNQRTCAEX
71 72
XSVELIHTMPCRKADG
73
D
HSCI
KNYQRLGATXMVEP
74
G
T
X
P
VRIQWK
75
LXAE
F
N
S
I
T
C
76
XFMRVATPYNDCGEQKL
W
HSI
77 78 79 80 81 82 83 84 85 86 87 88 89 90 91
YCPREGWHMSFNTQ
I
KADL
XV
92 93
VHQMLYFDR
XTAEGPKWSINC
94 95
V
T
X
C
H
YR
I
W
S
GA
ELN
Q
P
D
K
F
9697 98 99
CXFMRVYTDEGQAIWNSKLPH
100
DCNYKWQISX
PM
GHTFLAE
V
R
101
LIKQVD
Y
W
E
102
YDQSMWNHCAEXPTKVLRG
I
F
103
YDQSMWNHCAEXPTKVLRG
I
F
104 105
0
1
2
3
4
bits
01
L
X
Y
23456789
10 11 12 13 14 15 16 1718 19
M
W
GYQHSE
CKTDV
PI
ALF
RNX
20 21 22 23 24 25 26 2728 29 30 31 32 33 34 35 36 37 38 39 40 41 42 4344 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
LIRGFTQXSAEN
75
H
S
PL
KCDGYTEQMRI
NAVFX
76 77 78 79 80 81 82 83 8485 8687
S
G
L
8889 9091 92 9394 95 9697 98 99
100 101
TL
V
Q
X
102 103 104 105
Logos from the same data randomized through permutation
Features with 0.5 bits are significant at the 1% level
Function Logos of Bacterial tRNAs from tRNAdb
0
1
2
3
4
bit
s
5′
01
I
QW
2
V
E
RK
3
L
G
K
I
D
R
V
Y
S
4
V
F
L
G
T
X
M
I
R
P
W
S
C
H
N
5
I
F
R
H
V
N
C
A
Q
G
M
Y
L
P
T
S
6
X
D
HC
R
F
A
I
T
W
S
L
K
N
G
P
V
7
K
N
L
E
P
V
D
Y
H
RX
FT
IA
W
G
M
S
C
Q
89
X
Q
S
H
Y
P
D
C
G
T
W
N
R
I
V
F
A
M
K
10
L
G
C
V
E
11
R
E
Q
DX
12
G
S
C
1314
RL
D
E
S
H
Y
Q
C
15 1617
I
A
D
E
K
N
T
Q
R
G
W
L
18
R
K
T
S
CW
E
19
L
S
P
N
W
R
T
20 212223
L
G
D
CR
24
P
N
YL
25
R
EY
26 27
C
N
D
P
G
R
I
S
X
Y
W
T
V
F
E
A
H
Q
M
K
28
I
RT
P
G
D
E
H
Q
Y
S
L
C
29
Y
SP
D
H
G
W
T
R
I
V
F
A
K
N
M
30
T
R
Y
S
H
N
Q
L
W
G
D
E
31S
32 33
A
I
Y
G
P
C
L
S
W
R
T
K
F
N
Q
34
A
H
R
K
N
V
E
P
L
G
W
S
T
F
M
C
I
35
I
AT
M
S
C
K
F
D
P
X
W
R
Q
E
V
H
Y
L
G
36
L
I
RC
37
G
AW
EI
M
S
C
N
R
T
Q
L
K
H
F
Y
38
I
L
P
X
V
A
T
39 40
L
T
PR
41
X
V
I
F
M
L
42
KL
S
F
Y
C
W
43
H
L
Q
R
FG
YI
T
V
W
S
X
D
C
A
E
N
M
K
44
D
E
HM
A
VL
C
P
F
G
R
S
W
Y
T
I
X
N
K
45
T
I
V
R
D
Y
P
F
QS
C
W
46
SG
47
I
W
L
M
S
F
G
E
H
Y
N
R
Q
T
K
P
V
D
A
48
S
W
G
C
I
H
A
F
Q
T
X
M
K
V
R
P
N
L
49
T
I
A
L
E
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Adenine
Cytosine
Guanine
Uracil
gap
Freyhult et al. (2006) Nucleic Acids Research 34(3):915
Logos from the same data mutated in silico 5%
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N
80
PARSVENIHKWGLTYCFM
81
STI
P
E
8283
ISTRQ
84 85
YQKNCGEFRWVDSHM
P
T
L
I
X
8687
LWIADVNGHMTKRQCXEP
S
Y
88
S
A
V
D
C
89 90
GTLPVIYS
M
91
TLX
K
N
Y
Q
V
I
92
FYAPXWVRILKSQMGNCTH
93
ARTI
G
N
S
C
94
GLDWPYRI
S
N
C
T
A
K
V
E
95
SEFVKNDRMLCAW
P
T
G
I
H
Q
96
SGNQAITLVEKCFRHD
97
LEYWSGHTQPDRNVIMF
K
98
LRAE
K
S
V
Y
C
F
T
D
99
ISTLAVKFDM
N
R
E
H
100
ILRPVW
CN
X
Q
101
SDQNRTE
V
K
A
M
X
P
I
L
Y
F
102
ATHCK
103 104 105
SIAFDMK
0
1
2
3
4
bit
s
01
LRF
YN
Q
2
SGLFQM
V
K
N
R
E
H
3
IGPRLXMS
V
E
Y
C
F
T
D
4
XAEGYQLHSTDPVRIWMNFK
5
PAQWGTSCENLFVK
R
D
I
H
6
SVEYRKDCNLAFPWMTGI
H
Q
7
GFDWLPYRIS
C
N
T
A
K
V
E
89
IGRQHMEY
10
IRL
K
W
M
G
Q
S
11
TRYN
S
Q
H
L
G
D
W
E
12
YSPDHG
K
M
I
R
W
T
F
V
A
N
13
S
R
L
W
14
SIRKFYHT
P
W
Q
G
D
E
151617
S
A
R
T
I
E
18
EAPQVRXMIKWCDTSYGNFLH
19
SYCQGNWHFPTRXDKLVI
M
A
20
I
S
A
K
D
W
GX
P
21
SARGTLNMW
2223
AYEVSCMLTKGQWPDNHFXI
24
LTKNAXPVGC
Y
E
H
S
D
R
I
M
25
RGTID
Y
M
S
C
E
2627
GX
L
D
Q
C
28
LSAR
Q
Y
G
D
29
LA
R
T
K
Y
G
C
S
30
V
R
E
T
Q
M
F
DX
31
LISTKNYQWCEG
H
P
V
R
D
32
LIPSEFDGVY
N
33
LAIDPYEFSVKWHRCM
G
X
34
WGCSIHAFQXKTMV
R
N
P
L
35
IXWLMSCFEGHYNRQT
P
K
V
A
D
36
S
L
X
I
P
G
37
LAGTVRDIYPMF
Q
S
C
W
38
XDCSREFIVG
L
T
M
Q
A
P
H
3940
IFHWR
S
T
L
P
G
V
K
Q
A
E
41
ITSXH
Y
N
K
E
D
Q
42
LGQS
R
K
T
I
N
X
M
43
IASGDF
44
TIRGAE
M
P
V
L
H
Q
45
PWAXEMISCNRT
Q
H
L
K
F
Y
46
ARIXGDLWSPQC
47
IADMCKFTSXPWR
Q
E
V
H
Y
L
G
48
AHKNQVREPLW
G
T
S
M
F
C
I
49
AXVDGPLIYRSWTFQ
K
N
C
50
PVRDAITWMG
K
N
Q
H
C
Y
L
S
E
51
LPEKIQSRAVGMC
H
N
W
F
52
LXTWH
535455 56
GTVNQY
L
S
57
XVQSY
L
5859 6
0
6
1
TP
E
YS
6
2
6
3
GTDQWSLY
6
4
6
5
6
6
6
7
6
8
6
9
70 71
GQLKNS
72
AXKSYL
73 74
IVFKGSE
L
C
H
Q
Y
75
LEQGCWXYTRPDKVMHNFAI
76
AIXEFMK
N
D
T
Q
R
G
W
L
77
XS
VKYI
Q
WARPC
78
LISVRKTXMGAPNWCHQ
79
ATLHK
F
Y
X
Q
N
V
I
80
P
K
D
L
Q
I
81 828384
LRPAEDWCGQ
858687
LPRNQECKDV
M
T
W
G
H
A
F
88
AVHNKMDTQGCSLRWEIXYPF
89
LAX
E
90
XAREDQHSVNIKGTWLYFCM
91
AEFMSCLVGYTKIX
N
P
W
R
Q
92
EHNTGQIXPRLAFKVWMYS
93
KFSMWNXILCRPVTAGDEH
94
DVL
R
XHTYAIFWGCSMQ
95
DYQRHXCFAITWS
K
L
G
N
P
V
96
FDIWRVNHQ
A
C
M
G
Y
L
P
T
S
97
AVKFYRLXMIWTSCGPDNH
98
XEFNPLG
K
I
D
R
V
Y
S
A
99
A
V
G
W
R
DE
K
100
ASGKIQCRNL
X
E
M
W
101
A
N
ST
C
G
102
GTLPSED
103
A
G
I
104
LAPEQRF
Y
105
ARSGFNY
0
1
2
3
4
bit
s
0
ILARP
N
Q
CH
1
NWFAELYISMGKRVCDHT
2
GTLNRKEFVMQPISWDXAYC
3
FNSDYLV
R
K
I
G
Q
P
H
A
W
4
ERINFVDLPMWHTYSXQGCA
5
KIRDNHFTASCQPLYGVMWX
6
IGPATQRVNHCL
E
D
K
M
S
F
X
Y
7
EVACTSQIWRNFGMYDXLPH
89
RGIWT
Y
Q
C
D
E
P
H
X
S
L
10
M
CQGLEVW
NDTFKAPIRXH
1112
ISRTGKWE
Q
Y
H
P
D
X
L
13
R
G
E
I
14
IGV
F
H
CY
S
L
15 16 17
WGRQETINKMHADSPXYFCV
18
ALPR
E
F
G
H
T
M
K
D
I
19 20
RGLKFTNSC
21 2223
ST
L
Y
C
E
KV
A
2425
LS
26
L
T
S
A
R
27
I
S
T
F
P
C
NL
28
QDHEGWPTKRNVXIAFM
29
LTIGDWHPY
Q
S
E
C
30
EDGWLQSHNMFRTYIVAKPC
31
IRTE
L
K
M
C
S
Y
32
CEHGQPTNWVRILAYDS
M
X
33
PVRQHIWLS
T
K
F
N
C
Y
34
STPYNDIRELQ
G
M
F
C
35
GDPMVAITNKRYLQSHEXFWC
36 37
SGKDYVALEIHCQ
R
P
N
M
X
38
LSATDRPFW
39 40
ARLPGVTSI
F
C
H
N
Y
D
41
RLHS
A
P
T
42
SIVAKFYL
R
H
Q
P
43
IAXY
F
R
Q
L
H
P
44
IP
S
V
A
F
T
L
G
45
FMHKLPS
R
T
I
E
A
V
D
G
46
NALKMRS
P
G
I
D
V
T
E
47
AGLVEFKDP
T
R
S
N
M
I
48
E
X
HA
Y
D
49
FKNQWXRSTGHLIPVMA
E
D
50
LQHSENYTRIMGDWACVKP
F
51
FHEYSLWGNAV
M
D
R
K
I
T
P
X
52
R
F
S
53
LI
X
V
D
S
M
54
LSQ
E
55
AVTS
Y
L
56 57
TXEFYL
S
58
RGKDYQCLS
59
IRVAEKFDLS
6
0
6
1
6
2
RTY
L
S
6
3
6
4
6
5
6
6
6
7
6
8
6
9
70
IGTEKYCMLS
71
RXS
L
Y
72
GTS
Y
L
73 74
CEQSDHGLFPKRNIVTAXMW
75 76
RTV
K
D
I
E
C
77
ECAQVTKRNIYFLMSGXPWDH
78
GCNHQKLPXSMTWREYVIAF
79
IVCFQXAYTRPKLG
M
E
W
H
S
D
8081 8283
LS
T
P
A
E
Q
G
84
SL
I
P
V
8586 87
SFPXAHTQGR
M
K
V
W
D
N
I
L
88 89
IGPR
K
L
D
90
IA
G
T
R
S
91
SPKRHEGINY
X
L
V
T
A
C
F
92
SAMYHIKTWRVLEQGNPXCD
93
LPSEFNTI
Y
C
K
Q
94
IGE
F
T
S
LK
95
YKLHGQSPIVFNMTDWCAER
96
GYSLMTPVWQCAH
F
D
R
N
I
K
E
97
SGAPQWTFYL
N
V
K
D
I
R
E
98
GIYQVDRKL
T
F
C
E
X
M
N
99
AIXWQV
E
K
F
H
R
N
L
G
T
100
R
L
N
S
I
TP
101
VKE
R
N
S
D
W
Q
102 103 104 105
0
1
2
3
4
bit
s
0
SL
A
R
F
Q
1
RVGLNI
S
A
E
FX
P
2
PIQWMVE
K
F
H
R
N
L
G
T
3
SAGIYQDRKL
T
F
C
X
E
N
M
4
SAGTNPYQF
L
K
V
D
I
R
E
5
LPSVGYTWHCQA
I
R
F
D
N
K
E
6
XYHKLGSIQPMTNVDWA
E
C
R
7
I
S
F
T
D
X
L
K
89
ISTN
Y
W
GQ
E
10
LGP
F
T
NM
CS
Y
11
DWGLQHSNRYPMTAICFKV
12
IRGTXHPY
Q
S
E
C
1314
LSQHDGWPFT
R
I
K
V
X
A
M
N
15
STIPRKW
1617
ISRTLGM
18
LFGWNIKSCTDYMERXPVQA
19
AGDIHMREKTWLNS
P
X
F
20
G
P
F
D
S
X
2122
LRIPY
23
IXDAKNPWGQ
T
L
C
M
S
E
Y
24
LGPXARTNDIV
C
S
E
Y
25
TIR
K
Y
E
26
L
T
A
M
S
C
27
LRTIXQAE
28
GP
T
N
WY
29
ASTVRFWE
Q
Y
H
D
P
L
X
30
I
L
F
Q
H
E
S
31
SYDCRKPGVMHETLWQINFAX
32
RTLIA
D
Q
C
G
H
33
FCKNXQRWTGSMHLVIPE
D
A
34
MCILFNRPTGVKWQSEXHADY
35
GXFLEKP
T
S
R
N
M
I
36
LNQACRKMI
S
P
G
D
V
T
E
37
XFHCKLPS
R
T
I
E
A
V
D
G
38
AQMTLGFVIERSWDCXYNK
3940
VAGESPTIR
Q
K
L
W
X
M
41
ILAVHF
SC
R
G
W
42
ISLTKA
V
D
G
E
43
SRI
X
A
E
T
W
G
44
GTLIKYWSFV
H
C
Q
A
M
E
D
45
STLYAVEKIHCRQ
P
N
M
X
4647
VGMTDAPNIRYKLHQSEFWXC
48
ITYQWN
R
E
L
M
G
C
F
49
AGPRIQWSHCNLTKF
Y
50
KGTMVRICEW
S
N
Y
L
H
Q
51
IRG
W
Y
52
IALXSWH
53
SRX
T
L
D
5455 56
XDSFL
Y
57
RVKNTSL
Y
58
ARGPYL
S
596
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
LIRVC
S
6
9
IFKQ
LS
70
IGAYWLS
71
ITL
Y
S
7273
ARPFNDS
Y
L
74
RTPEKL
C
S
Q
F
H
75
GS
V
W
R
D
P
T
X
76
WGRQTMDECNKIXFAVSYPH
77
LSEFNHPTI
Y
K
C
Q
78
SQWMYHKIRPTVXLCEGND
79
PKQREGNIX
Y
L
V
T
F
A
C
80
GTSAEQRC
81
L
R
K
I
M
828384 85
IRGVTP
86
ILXSRE
87
SGEA
M
K
L
I
F
C
T
R
88
ISREWLDG
Q
T
M
K
N
H
V
A
899091
IVCFNXYTARWPKLG
E
S
M
H
D
92
VNWHGKQLXPMSTR
Y
I
E
A
F
93
EDGTCAVPQIRLNKSXFWMY
94
EVTACSQINGYMWRFLXHPD
95
IARTQHGPVNCL
E
D
M
K
S
F
Y
X
96
KRIHDNTAFCSQLPVGYMWX
97
IDRENVFHLPTMYWCQGS
A
X
98
ASXDYL
V
K
R
I
G
P
Q
H
W
99
GRLENKFVQDMWXAIP
S
Y
C
100
XNWMELCRISTKVGAYHFD
101
LGIS
V
A
T
D
W
MH
102 103 104
IGKDM
105
0
1
2
3
4
bit
s
0123
ARTLQX
4
R
I
Y
X
56
R
T
Q
X
7
LRIFW
8
IK
G
9
I
K
F
X
Y
1011 1213 14 15 1617
GLNQH
18 19
IMPLVTRKDWNHG
S
Q
E
Y
C
20
XSGFDKCHAWQNTILRYEMV
21
P
A
G
22
E
G
23
LSGTA
H
24
ILRMSDHCGPVN
A
X
T
K
W
F
Q
25
Y
SECRMIDTKPLGVAXWNHFQ
26 27 28
IRVGC
29 303132 3334 35 36 37 38 39 40 41
LRI
H
42
L
I
X
F
T
D
A
43
LAPVWHT
44 45
I
X
L
H
46
A
X
R
47
LAKHME
48 49
I
F
Q
W
50
IV
K
T
51
IXVRFKNHGTA
D
S
C
L
Y
52 53 5455 56 57 5859 60 6162 6364 65 6667 68 6970 71 7273 74
I
R
G
P
A
Q
C
S
75
AIPRYTMW
C
Q
G
E
S
L
7677
LXEFY
78
AXRGNW
79
IGLVF
8081 82
AIEY
Q
H
83 84 8586 87
IGV
D
W
A
88 89
R
T
I
P
G
90 91 9293
A
S
V
G
94 95
P
A
T
N
Y
96
IRGXS
97
LAC
98 99
GIPDXMKQ
100 101 102 103 104 105
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
Adenine
Cytosine
Guanine
Uracil
gap
0
1
2
3
4
bit
s
01
L
A
SI
QW
2
SAG
V
E
D
RK
3
AHPL
G
I
K
R
D
V
Y
S
4
AVFLGMRTIXWSPCHN
5
IFRHVNC
Q
A
G
Y
M
L
P
T
S
6
XDHCRAFTIWS
L
K
N
G
P
V
7
KLNVEPDYHXRIFTAWMGSCQ
8
Q
X
P
9
XQSHYPDCTGWIMRNKFAV
10
ISGALDMCVE
11
LIAGR
T
Y
Q
M
P
E
DX
12
IF
Q
M
G
C
S
1314
ITPRFL
D
E
S
H
Y
Q
C
15 1617
IADNE
K
T
Q
R
G
W
L
18
GIR
K
T
S
C
W
E
19
LSGIPE
Y
W
R
N
T
20 212223
L
I
A
G
V
K
D
CR
24
I
G
P
S
R
N
YL
25
L
R
P
S
EY
26 27
CQNXPDAEIYTFRSGWVHKM
28
IRXTWPGKD
E
H
Q
Y
C
S
L
29
LYSPDHG
V
W
F
R
T
K
A
I
M
N
30
ITRMYS
H
N
Q
L
W
D
G
E
31
T
P
FS
32
W
TPCEFK
33
ADIPCGYLSWT
R
K
F
N
Q
34
AHRKPVENLS
G
T
F
W
M
C
I
35
AFITMCKDPSWXQ
E
R
V
H
Y
L
G
36
LIGXRC
37
GAWEIMCSNR
T
Q
L
H
K
F
Y
38
GLI
X
V
F
Q
M
E
A
T
39
ATLFNMV
40
I
S
T
L
F
G
N
PR
41
ASGPCF
L
X
I
V
M
42
AGPVEKTNHL
S
F
Y
C
W
43
HLQRFGTWYAXIDEVSKCNM
44
DHMEAVLCFGPSWYITRKXN
45
TIAVDYRF
Q
PS
C
W
46
L
A
S
V
FG
47
IWSMLFGEHYNRQ
T
P
K
V
D
A
48
DYWSCGFIHAQTXMKVRP
N
L
49
AITLEFCPKVMWSYH
R
G
X
50
LSKFHEPWN
V
G
M
I
A
D
R
T
X
51
RAKDVW
G
S
C
H
E
Q
52
LRT
I
P
535455
IRTLXYS
56
GKXSL
57 58
ASFNYVL
59 6
0
6
1
6
2
6
3
IGTKL
S
6
4
IFDNHY
L
S
6
5
ATY
L
S
6
6
6
7
6
8
6
9
7071 72
IXVY
L
S
73
AVL
S
74
TXVKIRLNP
S
G
C
D
E
75
AXSREHY
76
S
RT
M
77
PWKDXSFLIGMCTR
N
V
A
E
78
XEFWRGTAIHPQ
L
M
Y
K
S
79
AESLWVGTMY
K
I
X
P
Q
R
N
80
PARSVENIHKWGLTYCFM
81
STI
P
E
8283
ISTRQ
84 85
YQKNCGEFRWVDSHM
P
T
L
I
X
8687
LWIADVNGHMTKRQCXEP
S
Y
88
S
A
V
D
C
89 90
GTLPVIYS
M
91
TLX
K
N
Y
Q
V
I
92
FYAPXWVRILKSQMGNCTH
93
ARTI
G
N
S
C
94
GLDWPYRI
S
N
C
T
A
K
V
E
95
SEFVKNDRMLCAW
P
T
G
I
H
Q
96
SGNQAITLVEKCFRHD
97
LEYWSGHTQPDRNVIMF
K
98
LRAE
K
S
V
Y
C
F
T
D
99
ISTLAVKFDM
N
R
E
H
100
ILRPVW
CN
X
Q
101
SDQNRTE
V
K
A
M
X
P
I
L
Y
F
102
ATHCK
103 104 105
SIAFDMK
0
1
2
3
4
bit
s
01
LRF
YN
Q
2
SGLFQM
V
K
N
R
E
H
3
IGPRLXMS
V
E
Y
C
F
T
D
4
XAEGYQLHSTDPVRIWMNFK
5
PAQWGTSCENLFVK
R
D
I
H
6
SVEYRKDCNLAFPWMTGI
H
Q
7
GFDWLPYRIS
C
N
T
A
K
V
E
89
IGRQHMEY
10
IRL
K
W
M
G
Q
S
11
TRYN
S
Q
H
L
G
D
W
E
12
YSPDHG
K
M
I
R
W
T
F
V
A
N
13
S
R
L
W
14
SIRKFYHT
P
W
Q
G
D
E
151617
S
A
R
T
I
E
18
EAPQVRXMIKWCDTSYGNFLH
19
SYCQGNWHFPTRXDKLVI
M
A
20
I
S
A
K
D
W
GX
P
21
SARGTLNMW
2223
AYEVSCMLTKGQWPDNHFXI
24
LTKNAXPVGC
Y
E
H
S
D
R
I
M
25
RGTID
Y
M
S
C
E
2627
GX
L
D
Q
C
28
LSAR
Q
Y
G
D
29
LA
R
T
K
Y
G
C
S
30
V
R
E
T
Q
M
F
DX
31
LISTKNYQWCEG
H
P
V
R
D
32
LIPSEFDGVY
N
33
LAIDPYEFSVKWHRCM
G
X
34
WGCSIHAFQXKTMV
R
N
P
L
35
IXWLMSCFEGHYNRQT
P
K
V
A
D
36
S
L
X
I
P
G
37
LAGTVRDIYPMF
Q
S
C
W
38
XDCSREFIVG
L
T
M
Q
A
P
H
3940
IFHWR
S
T
L
P
G
V
K
Q
A
E
41
ITSXH
Y
N
K
E
D
Q
42
LGQS
R
K
T
I
N
X
M
43
IASGDF
44
TIRGAE
M
P
V
L
H
Q
45
PWAXEMISCNRT
Q
H
L
K
F
Y
46
ARIXGDLWSPQC
47
IADMCKFTSXPWR
Q
E
V
H
Y
L
G
48
AHKNQVREPLW
G
T
S
M
F
C
I
49
AXVDGPLIYRSWTFQ
K
N
C
50
PVRDAITWMG
K
N
Q
H
C
Y
L
S
E
51
LPEKIQSRAVGMC
H
N
W
F
52
LXTWH
535455 56
GTVNQY
L
S
57
XVQSY
L
5859 6
0
6
1
TP
E
YS
6
2
6
3
GTDQWSLY
6
4
6
5
6
6
6
7
6
8
6
9
70 71
GQLKNS
72
AXKSYL
73 74
IVFKGSE
L
C
H
Q
Y
75
LEQGCWXYTRPDKVMHNFAI
76
AIXEFMK
N
D
T
Q
R
G
W
L
77
XS
VKYI
Q
WARPC
78
LISVRKTXMGAPNWCHQ
79
ATLHK
F
Y
X
Q
N
V
I
80
P
K
D
L
Q
I
81 828384
LRPAEDWCGQ
858687
LPRNQECKDV
M
T
W
G
H
A
F
88
AVHNKMDTQGCSLRWEIXYPF
89
LAX
E
90
XAREDQHSVNIKGTWLYFCM
91
AEFMSCLVGYTKIX
N
P
W
R
Q
92
EHNTGQIXPRLAFKVWMYS
93
KFSMWNXILCRPVTAGDEH
94
DVL
R
XHTYAIFWGCSMQ
95
DYQRHXCFAITWS
K
L
G
N
P
V
96
FDIWRVNHQ
A
C
M
G
Y
L
P
T
S
97
AVKFYRLXMIWTSCGPDNH
98
XEFNPLG
K
I
D
R
V
Y
S
A
99
A
V
G
W
R
DE
K
100
ASGKIQCRNL
X
E
M
W
101
A
N
ST
C
G
102
GTLPSED
103
A
G
I
104
LAPEQRF
Y
105
ARSGFNY
0
1
2
3
4
bit
s
0
ILARP
N
Q
CH
1
NWFAELYISMGKRVCDHT
2
GTLNRKEFVMQPISWDXAYC
3
FNSDYLV
R
K
I
G
Q
P
H
A
W
4
ERINFVDLPMWHTYSXQGCA
5
KIRDNHFTASCQPLYGVMWX
6
IGPATQRVNHCL
E
D
K
M
S
F
X
Y
7
EVACTSQIWRNFGMYDXLPH
89
RGIWT
Y
Q
C
D
E
P
H
X
S
L
10
M
CQGLEVW
NDTFKAPIRXH
1112
ISRTGKWE
Q
Y
H
P
D
X
L
13
R
G
E
I
14
IGV
F
H
CY
S
L
15 16 17
WGRQETINKMHADSPXYFCV
18
ALPR
E
F
G
H
T
M
K
D
I
19 20
RGLKFTNSC
21 2223
ST
L
Y
C
E
KV
A
2425
LS
26
L
T
S
A
R
27
I
S
T
F
P
C
NL
28
QDHEGWPTKRNVXIAFM
29
LTIGDWHPY
Q
S
E
C
30
EDGWLQSHNMFRTYIVAKPC
31
IRTE
L
K
M
C
S
Y
32
CEHGQPTNWVRILAYDS
M
X
33
PVRQHIWLS
T
K
F
N
C
Y
34
STPYNDIRELQ
G
M
F
C
35
GDPMVAITNKRYLQSHEXFWC
36 37
SGKDYVALEIHCQ
R
P
N
M
X
38
LSATDRPFW
39 40
ARLPGVTSI
F
C
H
N
Y
D
41
RLHS
A
P
T
42
SIVAKFYL
R
H
Q
P
43
IAXY
F
R
Q
L
H
P
44
IP
S
V
A
F
T
L
G
45
FMHKLPS
R
T
I
E
A
V
D
G
46
NALKMRS
P
G
I
D
V
T
E
47
AGLVEFKDP
T
R
S
N
M
I
48
E
X
HA
Y
D
49
FKNQWXRSTGHLIPVMA
E
D
50
LQHSENYTRIMGDWACVKP
F
51
FHEYSLWGNAV
M
D
R
K
I
T
P
X
52
R
F
S
53
LI
X
V
D
S
M
54
LSQ
E
55
AVTS
Y
L
56 57
TXEFYL
S
58
RGKDYQCLS
59
IRVAEKFDLS
6
0
6
1
6
2
RTY
L
S
6
3
6
4
6
5
6
6
6
7
6
8
6
9
70
IGTEKYCMLS
71
RXS
L
Y
72
GTS
Y
L
73 74
CEQSDHGLFPKRNIVTAXMW
75 76
RTV
K
D
I
E
C
77
ECAQVTKRNIYFLMSGXPWDH
78
GCNHQKLPXSMTWREYVIAF
79
IVCFQXAYTRPKLG
M
E
W
H
S
D
8081 8283
LS
T
P
A
E
Q
G
84
SL
I
P
V
8586 87
SFPXAHTQGR
M
K
V
W
D
N
I
L
88 89
IGPR
K
L
D
90
IA
G
T
R
S
91
SPKRHEGINY
X
L
V
T
A
C
F
92
SAMYHIKTWRVLEQGNPXCD
93
LPSEFNTI
Y
C
K
Q
94
IGE
F
T
S
LK
95
YKLHGQSPIVFNMTDWCAER
96
GYSLMTPVWQCAH
F
D
R
N
I
K
E
97
SGAPQWTFYL
N
V
K
D
I
R
E
98
GIYQVDRKL
T
F
C
E
X
M
N
99
AIXWQV
E
K
F
H
R
N
L
G
T
100
R
L
N
S
I
TP
101
VKE
R
N
S
D
W
Q
102 103 104 105
0
1
2
3
4
bit
s
0
SL
A
R
F
Q
1
RVGLNI
S
A
E
FX
P
2
PIQWMVE
K
F
H
R
N
L
G
T
3
SAGIYQDRKL
T
F
C
X
E
N
M
4
SAGTNPYQF
L
K
V
D
I
R
E
5
LPSVGYTWHCQA
I
R
F
D
N
K
E
6
XYHKLGSIQPMTNVDWA
E
C
R
7
I
S
F
T
D
X
L
K
89
ISTN
Y
W
GQ
E
10
LGP
F
T
NM
CS
Y
11
DWGLQHSNRYPMTAICFKV
12
IRGTXHPY
Q
S
E
C
1314
LSQHDGWPFT
R
I
K
V
X
A
M
N
15
STIPRKW
1617
ISRTLGM
18
LFGWNIKSCTDYMERXPVQA
19
AGDIHMREKTWLNS
P
X
F
20
G
P
F
D
S
X
2122
LRIPY
23
IXDAKNPWGQ
T
L
C
M
S
E
Y
24
LGPXARTNDIV
C
S
E
Y
25
TIR
K
Y
E
26
L
T
A
M
S
C
27
LRTIXQAE
28
GP
T
N
WY
29
ASTVRFWE
Q
Y
H
D
P
L
X
30
I
L
F
Q
H
E
S
31
SYDCRKPGVMHETLWQINFAX
32
RTLIA
D
Q
C
G
H
33
FCKNXQRWTGSMHLVIPE
D
A
34
MCILFNRPTGVKWQSEXHADY
35
GXFLEKP
T
S
R
N
M
I
36
LNQACRKMI
S
P
G
D
V
T
E
37
XFHCKLPS
R
T
I
E
A
V
D
G
38
AQMTLGFVIERSWDCXYNK
3940
VAGESPTIR
Q
K
L
W
X
M
41
ILAVHF
SC
R
G
W
42
ISLTKA
V
D
G
E
43
SRI
X
A
E
T
W
G
44
GTLIKYWSFV
H
C
Q
A
M
E
D
45
STLYAVEKIHCRQ
P
N
M
X
4647
VGMTDAPNIRYKLHQSEFWXC
48
ITYQWN
R
E
L
M
G
C
F
49
AGPRIQWSHCNLTKF
Y
50
KGTMVRICEW
S
N
Y
L
H
Q
51
IRG
W
Y
52
IALXSWH
53
SRX
T
L
D
5455 56
XDSFL
Y
57
RVKNTSL
Y
58
ARGPYL
S
596
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
LIRVC
S
6
9
IFKQ
LS
70
IGAYWLS
71
ITL
Y
S
7273
ARPFNDS
Y
L
74
RTPEKL
C
S
Q
F
H
75
GS
V
W
R
D
P
T
X
76
WGRQTMDECNKIXFAVSYPH
77
LSEFNHPTI
Y
K
C
Q
78
SQWMYHKIRPTVXLCEGND
79
PKQREGNIX
Y
L
V
T
F
A
C
80
GTSAEQRC
81
L
R
K
I
M
828384 85
IRGVTP
86
ILXSRE
87
SGEA
M
K
L
I
F
C
T
R
88
ISREWLDG
Q
T
M
K
N
H
V
A
899091
IVCFNXYTARWPKLG
E
S
M
H
D
92
VNWHGKQLXPMSTR
Y
I
E
A
F
93
EDGTCAVPQIRLNKSXFWMY
94
EVTACSQINGYMWRFLXHPD
95
IARTQHGPVNCL
E
D
M
K
S
F
Y
X
96
KRIHDNTAFCSQLPVGYMWX
97
IDRENVFHLPTMYWCQGS
A
X
98
ASXDYL
V
K
R
I
G
P
Q
H
W
99
GRLENKFVQDMWXAIP
S
Y
C
100
XNWMELCRISTKVGAYHFD
101
LGIS
V
A
T
D
W
MH
102 103 104
IGKDM
105
0
1
2
3
4
bit
s
0123
ARTLQX
4
R
I
Y
X
56
R
T
Q
X
7
LRIFW
8
IK
G
9
I
K
F
X
Y
1011 1213 14 15 1617
GLNQH
18 19
IMPLVTRKDWNHG
S
Q
E
Y
C
20
XSGFDKCHAWQNTILRYEMV
21
P
A
G
22
E
G
23
LSGTA
H
24
ILRMSDHCGPVN
A
X
T
K
W
F
Q
25
Y
SECRMIDTKPLGVAXWNHFQ
26 27 28
IRVGC
29 303132 3334 35 36 37 38 39 40 41
LRI
H
42
L
I
X
F
T
D
A
43
LAPVWHT
44 45
I
X
L
H
46
A
X
R
47
LAKHME
48 49
I
F
Q
W
50
IV
K
T
51
IXVRFKNHGTA
D
S
C
L
Y
52 53 5455 56 57 5859 60 6162 6364 65 6667 68 6970 71 7273 74
I
R
G
P
A
Q
C
S
75
AIPRYTMW
C
Q
G
E
S
L
7677
LXEFY
78
AXRGNW
79
IGLVF
8081 82
AIEY
Q
H
83 84 8586 87
IGV
D
W
A
88 89
R
T
I
P
G
90 91 9293
A
S
V
G
94 95
P
A
T
N
Y
96
IRGXS
97
LAC
98 99
GIPDXMKQ
100 101 102 103 104 105
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
Adenine
Cytosine
Guanine
Uracil
gap
Logos from the same data mutated in silico 20%
0
1
2
3
4
bit
s
01
LPSRTDYAMCEGIQHW
2
LGXSAEIWVRFTQMYK
3
AXHPML
G
K
I
R
D
V
Y
S
45
EDIVCNRXHKFGAQY
L
M
P
T
S
6
RQXCHAFDTISWN
L
K
G
V
P
7
NVEKPLRDXYTHFIMAGSCWQ
89
ELQXSHYPDGCNWTKRIVAMF
10 11
ISE
Q
W
L
R
DX
12
RIKQLHATNYPGMSC
13
XSQWALMDRVEHNC
14
ARPXKFNTMILD
E
S
H
Y
Q
C
15 16
IRLEFDQHSCATMNPY
17
KAIPVCESX
T
N
Q
R
G
W
L
18
IALXSYRGFPVTKCWE
19
ITPXSALNQWR
K
M
20
LRTIXPVDQASG
21
ISVNLRGXPFW
2223
L
E
F
G
N
Y
Q
W
A
H
X
D
CR
24
SGV
R
E
F
D
I
P
N
Y
X
T
WL
25
LGTIPVFSCREW
X
QY
26 27
C
N
Q
EDTSYHVGW
AIRPXFMK
28
PFAMIKTNXVGRWE
D
H
Y
Q
C
L
S
29
EQSLCYXPHDGKTN
V
I
R
M
F
W
A
30
IPAVNYRTFHS
Q
L
W
D
G
E
31
LXVKGDAC
I
Y
Q
H
S
32 33
VYAMPCEIGDLWRSTFKN
Q
34
AKQHNREPVGWLT
C
F
S
M
I
35
AWIXFSDNTKMRPE
Q
H
V
Y
L
G
36 37
EDWPVAXGIMSCRNTQ
F
L
H
K
Y
38
SIXPVELNRMAG
W
T
39
PRKFTDNYWLHMAVGQ
40
AVEKGDNIWSPC
L
T
M
R
41
AGSEDNQHX
M
I
L
V
F
42
TIPXKNADQL
S
C
W
Y
F
43
QLRFGITSKAYXMWDNVCE
44
DMEHAVLCPFXRSGIWYTKN
45
GLATDNYRIEKHPQF
S
C
W
46
IRFTNLYWHPVACESG
47
SIMXLGYWEHFRNQT
P
K
D
V
A
48
YGSIFWEHAQKXMRTVN
P
L
49
DAILETPYFVMWKSCRGHX
50
LSFCHPEVGWN
R
I
M
D
A
T
X
51
RXWTAKFDIGLNVSCH
E
Q
52
XPAVRTDWSGIENM
53
ILAVEKDRHMFYXPSN
54
VA
E
FLDIXMKH
55 5657 5859 6
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
6
9
7071 7273 74
IXFTYARLPMSN
G
D
C
E
75
LRDITAEYQXSVHF
76
LSGEWRTMPK
I
V
A
D
77
YWHMPDXKISLGFRNTCV
A
E
78
VFARTGHXQL
I
P
M
Y
K
S
79
SAECVFLGWMKYITX
Q
R
N
P
80
APXRDHSEWYVIKGLTNCMF
81
IXSANDRHGTPWEMV
8283
GLSTNDYQWP
I
F
84
GTIPLQWASC
R
X
85
ANYKQGFRDEWCSVHTLMPIX
8687
LWAIMDVNHTGKRQCXEY
S
P
88
RVLWAHGMS
D
Q
P
89 90
AGTXSEKDWICLQPN
91
ASKTRLGDPX
N
H
Y
Q
V
I
92
RIVGLNSQMKTCH
93
LAFDIRHTMSCGKQP
94
XQLHPGDRYWISNACT
E
K
V
95
FNYSVEKDRMCLPAWG
T
I
H
Q
96
APXNQGSCEKWVTLIRFHD
97
X
AGTPSEWRLHDYQVINMFK
98
IAGNRHMXKLESVYQFCT
D
99
XKFTDLYIGPMCSANREH
100
SPVEFGTDAMCLIRYWX
N
Q
101
DWSGQHRTEF
Y
L
I
X
K
A
V
M
P
102
TLGIACEKSDNXH
103 104 105
ILXENAGSCPT
H
Q
0
1
2
3
4
bit
s
0
RLNIASE
W
V
D
M
1
LIXSVKTYWGPAHN
Q
2
AGTIPXSFDLYMVKRNE
H
3
IGLXNRHMAQWSEYVFC
T
D
4
AQGSLYPTHIVDWRMNFK
5
GPSATYQCXNVLWEKDFIRH
6
XKSEDNYRVLCPAFWMTGIQH
7
GDLYQHMPRWSICA
T
N
K
E
V
89
LSRDIWGXENY
10
ALEGKNIVQPSFCM
11
AIXVTYRNH
S
Q
L
G
W
D
E
12
EYLXSPHDM
K
G
N
T
F
I
W
V
A
R
13
IASVFNLYWMPREDG
14
VTISALXMRYPW
Q
D
G
E
1516
LARGTVYIFDXH
17
SGVFDRLIXMATWECQ
18
EPAQVIRCXDMKSWTYGNLFH
19
SCEYQNGWHFPXTRDLKVMAI
20
IALVKNRE
T
S
D
G
W
X
P
21
SVGTYIA
L
E
D
H
P
22
IRKDYSTXPALENWH
23
YAVCESLTXMGQPNHKDFIW
24
XFNWAKTVPGC
Y
H
E
S
R
I
D
M
25
RLIPXVFKGTYWMANCSE
2627
RGEFLYQSXATWDC
28
ISAREKTLHXVCNGYD
29
AIVEFKLP
N
T
G
C
S
30
STPYQA
L
V
C
R
D
N
E
HX
31
LAKFTPMSCEGNVIHW
R
D
32
ALRFKIQSHXCEVGYN
33
TAPLIEYDVSKFWHMGCRX
34
SEGWIQATXMFHVKRLN
P
35
XISLWHFMYEGNQRP
T
K
V
D
A
36
XAREKGDLYHMIPF
37
LATGYIRPMVEQ
F
S
C
W
38
NYWXCSRDIEFVGLTMQP
A
H
3940
XIHCFWRST
L
V
G
P
K
Q
A
E
41
ARTXSLIVCN
Q
Y
D
H
K
E
42
LAVGQCEFDYPS
R
N
K
I
T
X
M
43
S
A
G
F
I
P
R
44
IXGTWEFDRSAVMP
L
H
Q
45
PWAGXEICSNMRTQ
K
H
L
F
Y
46
ARG
FWIXPVLSDECQ
47
IAMFDXPWTKQSER
V
H
L
Y
G
48
AXDRVKQEHPLGWTNS
M
F
C
I
49
PEDAXGLIYSTWRCFK
N
Q
50
XAIRTPDVWMGKQNCHL
E
Y
S
51
TLDQAPXIEKRSGVCNMHWF
52
GLIXAVEFWPMSTN
53
TPXSDLHCVRGFMK
54
REKTWSAIN
D
P
55 5657 58596
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
6
9
70 71727374
IARXKFTDNWGMSCLVEHQ
Y
75
LEQGWCXYFARTDKPVIHMN
76
XSVIAMFDK
N
T
R
G
Q
W
L
7778 79
RTWASHCKLQ
F
M
N
Y
X
V
I
8081
AGETQI
H
D
N
R
C
828384 85
PRKNDIYQWSLXAVTC
86
LGIPVTSARHYXWN
87
XPYSRLNQCEDKVTMWGA
H
F
88
G
DNLT
S
EWIX
RFYC
8990
XAENDWHSRVGKILTYCMF
91
SAFMCELVGTYIKP
X
N
Q
R
W
92
NHEQTGXIPRLMAFKVYWS
93
KSMWXFNILCVRPTAGDHE
9495
RYQMXCFAEHTIWSLK
G
V
P
N
96
XEFRIHVQAC
N
M
G
L
Y
T
P
S
97
KFYMALIWGRSXNTCPDH
98
PTLHXEGFNK
D
V
I
R
Y
S
A
99
PSVGTDRIHCFLAQWE
K
100
SVRGNITCAKFYQDXLHEMW
101
LRPAVDIQHX
M
S
K
NT
G
C
102
LPXFKYQWAHMCISNV
103
Y
104 105
LPXVARHGMIF
S
Y
0
1
2
3
4
bit
s
0
LTPVC
F
K
Y
Q
S
G
H
1
XPWNCGHSEIYAVLRTFKMD
2
GTRLNEKFVCYMIDSWQAXP
3
XMCETSDYLVKR
G
I
A
Q
P
H
W
4
IERKNVDFMWTPLHYCSQGXA
5
DIRFNQHATCSPLYWVGMX
6
PTIWRQVACNLEDK
M
S
F
X
Y
7
EVACSTQIWNMGFYDRPHXL
8
LRIVGFTSNY
9
IRAVGWTCY
Q
E
D
P
X
H
S
L
10
SYCMVKQGENTFLWRDAHIPX
1112
ATISVKWRMCGFE
Q
Y
D
P
H
L
X
13
LPAREKDYWIT
S
Q
14
AIPVFTDQWG
H
X
M
C
R
E
NY
S
L
15 16 17
GQRTNCEAXMSDPIVFKHY
18
VAG
P
S
C
R
E
D
L
H
K
T
I
19
LXRFYIQG
T
S
V
20 2122 23
RGIEKTY
Q
S
L
C
N
X
PV
A
24
TPAKGLDYWSHVRNI
25
AL
I
P
E
K
D
Q
G
T
M
YS
26
TPVKLQHXAIRESNDY
27
RF
T
Y
Q
I
S
V
C
G
N
PL
28
SYCQHDEGNRKTVXIWFPAM
29
AGTLVRKNIHMPDY
Q
S
E
C
30
XDWGQHLSFRANPVCYTIMK
31
RGLPEKFDNWAIVX
C
M
S
Y
32
FEGNHQVPTIWRLASYDMX
33
AGXPRELDQVIWHSNTCFKY
34
XKNYASHTEPWIFMRQLGC
35
VGMPDTAINRKYLSQEHFCWX
36 37
GTWEKFLYVADICRHQN
M
P
X
38
TIPRKDLGHXESWVQ
39 40
XEQMARLPVGTSI
C
H
N
Y
D
F
41
LREGYIXF
M
NS
A
P
T
42
SIVKFTNCDWGL
R
P
Q
H
43
ATIMKSG
Y
R
F
L
Q
H
P
44
PVRTNDYISAHXMELG
45
MCFWHPLKS
R
T
I
E
G
D
A
V
46
YWAHLNKCRSVIPG
T
D
M
E
47
LKDQWHVGFAEC
P
T
S
R
N
M
I
48 49
KFXNRQTSWHGMILVADEP
50
XQLENSHTMRIGDWACVKPF
51
HCQESLYWVGMKNAIDRX
P
T
52
RLPADYIWSTHM
5354
TXSFNYLIPREHC
55 56 575859 6
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
6
9
70 71 7273 74
EYCQSHDGLNMTVFPRKAIXW
75
GTISFNDRMCALYWQ
76
AVREKIYSHGTLNFC
77
CATQVRKNIFYGXWSLDMPH
78
DNGHCQXKLMTPSYWRIEAVF
79
NIVCAXTRFYQKPLMGWEHSD
80
MVFNCKYLWHTGIDPRXSEQA
81 82
ATLISKQXMRE
83
LARETWS
Y
F
G
Q
84
SXAVEKFYQWLTMRNI
8586
SEFT
D
Y
I
M
K
P
G
87
SPFHGAXRTQM
K
V
W
D
N
I
L
88
XVKFTQSL
I
G
P
A
89
GXAEFTDSIN
K
90
LSTDY
Q
A
I
R
G
M
N
91
REDQHPSGKNIYXL
V
T
A
F
C
92
ASYHIMVKRWTLQCEGPXND
93
GXEFWSHPMAILNTVYKCQ
94
VREFNQSAGXMPITLWK
95
YXKQLHPSGIFMTVDNEWACR
96
GLSPYVTWQHA
C
R
F
D
N
I
E
K
97
AXSGYWPTQF
L
K
N
D
V
I
R
E
98
SIPQWAHGVDYRKF
C
L
T
M
X
E
N
99
XPAYQSCDIWMKVE
F
H
N
R
L
G
T
100
TSRIYQA
L
H
X
M
W
E
KP
101
ATIXKGHFLE
R
S
Q
N
D
W
102 103
RLVAGYSHCEIQPTX
104 105
LTISVKNRGXEYWA
0
1
2
3
4
bit
s
0
EF
DYWS
R
LHTPANGC
1
IARTNYWLGV
C
S
FX
P
2
ISAQXYWVEK
C
F
H
N
R
L
T
G
3
AQSIGYDRKLTC
F
M
E
X
N
4
SWAHGQXNYPTFL
K
V
D
R
I
E
5
XLSYGVTPHWQFCARNIDE
K
6
XIGLHKMSQVPTDNAWCE
R
7
RGIXVENATSDPLFK
89
IAXPSVRFTLGNYCK
Q
E
10
IXATRLGHMC
E
D
V
K
S
Y
11
XEDWGLQSNRHTVYIPAKCFM
12
GLAVFNIHXPMKTWY
Q
C
E
S
13
RIXAKLDWHEGYSTV
14
EDHGP
W
X
M
T
R
V
A
I
N
F
K
151617 18
LGTCWNIKSYEDRMXVPAQ
19
GYMADIRHKSTLNWP
X
F
20
PEKTRIGSFDNQH
X
21
AVKFTSRLIXMPGY
22
IPXAFQWSREKLYC
23
IRXAFDWKNPHQGT
L
M
C
S
E
Y
24
AGPFLIWHTCRDV
S
E
Y
25
TIPSARYQMCGEK
26 2728
GSVLQTPCIFKNYRE
29
AVRIWTMCFSKGE
Q
Y
D
H
P
L
X
3031
Y
DMICRHWVENKXPAGTLQF
32
XARNWLITPSVEFQCGH
33
FKCQNXRGWTSHMDIVLE
P
A
3435
ALEKD
P
R
T
S
N
M
I
36
XFNLQAHKRMDPI
S
G
V
T
E
37
XFYCNHMLPKS
R
T
D
I
V
A
E
G
38
PAQTMLVGWEIFRCSDXNYK
39
APXSVRTNDHML
40
FDNCGYVHATSPEIRQLKW
M
X
41
LIQATPMVKYX
S
C
G
W
R
42
RLPKTWSIXMCD
G
A
E
V
43
SRFWAXG
L
D
I
Q
H
T
44
GTXRKNWISYLPFVCHQAM
E
D
45
ATDHGSVLKYECIRQN
M
P
X
4647
VGMTAIPNDRKLYSHQEFWCX
48
TXSAVEKNIPRDLMYGFC
49
RGPXKDQWHLASIVNTFC
Y
50
APXKFTMGNICWRE
S
H
Y
L
Q
51
LSVTQRGXAIEKYPW
52
SGVDLRHXAIYW
K
53
GTISREKAMVXWD
54
ASKDLYWRGXC
I
55 5657 58596
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
6
9
7071727374
IPVRFGNDYAEKSLCQH
75
GVFDYQARTSLWEKPX
76
QGRTDKHANCVIFESMXPY
77
VGFDLSRHMEXPITWKYCQ
78
SAWMYHKIRPTELVXGCND
79
SDQWHMPIGREXKNYLT
V
F
C
A
8081 8283
AGXREKTLHPMVISC
84 85
AVREKGTDLQWSIF
86
IRGXAEFSTCKVQ
87
SPXEDQWHMVAGFLIYCTR
88
SDIWRLPXGEQTK
N
H
M
A
V
899091
IQCFVNXAYTWRPKLGH
D
S
E
M
92
HXGVKMLSCQPTYR
E
I
F
A
93
ECGDATPVQRKNILFSXMWY
94
ECVATSWQINXFMGYHLRPD
95
ATNQWVGRPCLDKE
M
S
F
X
Y
96
KNDICTRHSFAQPLWYGVMX
97
DIREKNVFHLPMTCWQYSXG
A
98
ATXSFYDMCLR
K
V
I
Q
G
H
P
W
99
GLERKNFMDWACVISYPQX
100
XNWEMLYKHCRIADGTFVS
101
ALXETIQSRCGWFYH
102 103 104
ARPDNILHTX
K
M
105
0
1
2
3
4
bit
s
0
S
W
EFAVT
Q
YRMLPN
KIX
1
AKNLYWRGSVIXC
E
2
GKYASRPMEFVILD
C
3
XAREGFKLIPMTSY
456
GLS
F
T
D
I
R
M
A
78
GLXREFDIWHTSK
9
10
LREDYWAGCKST
N
11
STLIAENWRGPKV
12 13 14
IRXPKFGTQSEAVH
15
IX
V
L
W
T
S
AF
16 17
RIPEGNDYQSHMALW
18 19
FIMAPKLRTVWDNHGSQYEC
20
PXGSDWFHTVNKERQILAMYC
21
SAXVEFTDQGC
I
R
22
ILSRFKTDNYWXEVHG
23
RGPSKL
Y
A
X
I
V
24
DISRMLHVPWCGTN
X
K
F
A
Q
25
SYECQKGMXWTRPAILNFVDH
26 27
ATXSGDQLIRH
N
E
28
RXSKTNDLGY
H
29 30
R
H
SACGFDPW
31
SALXVKTDNHMPEI
32
VARELDYQTINWP
33
ARTXVELIPGNQC
34
RGPXNMEFKTLYA
C
35 36
ASGTVFLNRDXPH
37
PVENARLIXCFWD
38
SLXREKNYAMIPWFC
39
ARLVGKTNDIHCS
40 41
X
FIYLGPANDQW
42 43 4445 46
LPGKNQSIACRTHF
47
REFGKNQWSALPCTD
48
AXSVEQLHIKFTDGRC
4950
ALPVGTSMI
Y
R
X
K
51
TIXPVEFRMG
A
K
D
C
S
L
Y
52 53
T
54 55 56 57 58 596
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
6
9
70 7172 7374
RPXVFNIC
L
G
Y
Q
T
H
D
S
75
PAKFTDIRXY
W
Q
C
G
E
S
L
7677
SLPAGDWRMCIFNT
78
SNIAGMPLFYQTE
79
AGTPXDNIQSLHVRK
80
PSEFDWRXLKHMV
81 8283
XEKDL
I
G
S
A
W
M
84 85
LPXSFDYWAIGE
86 87
SEFKWRLTVAIGD
M
88 89
LTISEGYARCKQX
90
TLPEX
M
I
G
N
R
W
91
IATPXVFGNWSLEKD
92 93 94
LTEGKNQIMFSAXHW
95
PVGNLQRHXMITSAKD
96
IAKTDLRGPMSWEQ
97 98
SIEGDLARMKYXF
99
100
SAVKNIYRLGPCE
101 102 103 104 105
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
Adenine
Cytosine
Guanine
Uracil
gap
0
1
2
3
4
bit
s
01
LPSRTDYAMCEGIQHW
2
LGXSAEIWVRFTQMYK
3
AXHPML
G
K
I
R
D
V
Y
S
45
EDIVCNRXHKFGAQY
L
M
P
T
S
6
RQXCHAFDTISWN
L
K
G
V
P
7
NVEKPLRDXYTHFIMAGSCWQ
89
ELQXSHYPDGCNWTKRIVAMF
10 11
ISE
Q
W
L
R
DX
12
RIKQLHATNYPGMSC
13
XSQWALMDRVEHNC
14
ARPXKFNTMILD
E
S
H
Y
Q
C
15 16
IRLEFDQHSCATMNPY
17
KAIPVCESX
T
N
Q
R
G
W
L
18
IALXSYRGFPVTKCWE
19
ITPXSALNQWR
K
M
20
LRTIXPVDQASG
21
ISVNLRGXPFW
2223
L
E
F
G
N
Y
Q
W
A
H
X
D
CR
24
SGV
R
E
F
D
I
P
N
Y
X
T
WL
25
LGTIPVFSCREW
X
QY
26 27
C
N
Q
EDTSYHVGW
AIRPXFMK
28
PFAMIKTNXVGRWE
D
H
Y
Q
C
L
S
29
EQSLCYXPHDGKTN
V
I
R
M
F
W
A
30
IPAVNYRTFHS
Q
L
W
D
G
E
31
LXVKGDAC
I
Y
Q
H
S
32 33
VYAMPCEIGDLWRSTFKN
Q
34
AKQHNREPVGWLT
C
F
S
M
I
35
AWIXFSDNTKMRPE
Q
H
V
Y
L
G
36 37
EDWPVAXGIMSCRNTQ
F
L
H
K
Y
38
SIXPVELNRMAG
W
T
39
PRKFTDNYWLHMAVGQ
40
AVEKGDNIWSPC
L
T
M
R
41
AGSEDNQHX
M
I
L
V
F
42
TIPXKNADQL
S
C
W
Y
F
43
QLRFGITSKAYXMWDNVCE
44
DMEHAVLCPFXRSGIWYTKN
45
GLATDNYRIEKHPQF
S
C
W
46
IRFTNLYWHPVACESG
47
SIMXLGYWEHFRNQT
P
K
D
V
A
48
YGSIFWEHAQKXMRTVN
P
L
49
DAILETPYFVMWKSCRGHX
50
LSFCHPEVGWN
R
I
M
D
A
T
X
51
RXWTAKFDIGLNVSCH
E
Q
52
XPAVRTDWSGIENM
53
ILAVEKDRHMFYXPSN
54
VA
E
FLDIXMKH
55 5657 5859 6
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
6
9
7071 7273 74
IXFTYARLPMSN
G
D
C
E
75
LRDITAEYQXSVHF
76
LSGEWRTMPK
I
V
A
D
77
YWHMPDXKISLGFRNTCV
A
E
78
VFARTGHXQL
I
P
M
Y
K
S
79
SAECVFLGWMKYITX
Q
R
N
P
80
APXRDHSEWYVIKGLTNCMF
81
IXSANDRHGTPWEMV
8283
GLSTNDYQWP
I
F
84
GTIPLQWASC
R
X
85
ANYKQGFRDEWCSVHTLMPIX
8687
LWAIMDVNHTGKRQCXEY
S
P
88
RVLWAHGMS
D
Q
P
89 90
AGTXSEKDWICLQPN
91
ASKTRLGDPX
N
H
Y
Q
V
I
92
RIVGLNSQMKTCH
93
LAFDIRHTMSCGKQP
94
XQLHPGDRYWISNACT
E
K
V
95
FNYSVEKDRMCLPAWG
T
I
H
Q
96
APXNQGSCEKWVTLIRFHD
97
X
AGTPSEWRLHDYQVINMFK
98
IAGNRHMXKLESVYQFCT
D
99
XKFTDLYIGPMCSANREH
100
SPVEFGTDAMCLIRYWX
N
Q
101
DWSGQHRTEF
Y
L
I
X
K
A
V
M
P
102
TLGIACEKSDNXH
103 104 105
ILXENAGSCPT
H
Q
0
1
2
3
4
bit
s
0
RLNIASE
W
V
D
M
1
LIXSVKTYWGPAHN
Q
2
AGTIPXSFDLYMVKRNE
H
3
IGLXNRHMAQWSEYVFC
T
D
4
AQGSLYPTHIVDWRMNFK
5
GPSATYQCXNVLWEKDFIRH
6
XKSEDNYRVLCPAFWMTGIQH
7
GDLYQHMPRWSICA
T
N
K
E
V
89
LSRDIWGXENY
10
ALEGKNIVQPSFCM
11
AIXVTYRNH
S
Q
L
G
W
D
E
12
EYLXSPHDM
K
G
N
T
F
I
W
V
A
R
13
IASVFNLYWMPREDG
14
VTISALXMRYPW
Q
D
G
E
1516
LARGTVYIFDXH
17
SGVFDRLIXMATWECQ
18
EPAQVIRCXDMKSWTYGNLFH
19
SCEYQNGWHFPXTRDLKVMAI
20
IALVKNRE
T
S
D
G
W
X
P
21
SVGTYIA
L
E
D
H
P
22
IRKDYSTXPALENWH
23
YAVCESLTXMGQPNHKDFIW
24
XFNWAKTVPGC
Y
H
E
S
R
I
D
M
25
RLIPXVFKGTYWMANCSE
2627
RGEFLYQSXATWDC
28
ISAREKTLHXVCNGYD
29
AIVEFKLP
N
T
G
C
S
30
STPYQA
L
V
C
R
D
N
E
HX
31
LAKFTPMSCEGNVIHW
R
D
32
ALRFKIQSHXCEVGYN
33
TAPLIEYDVSKFWHMGCRX
34
SEGWIQATXMFHVKRLN
P
35
XISLWHFMYEGNQRP
T
K
V
D
A
36
XAREKGDLYHMIPF
37
LATGYIRPMVEQ
F
S
C
W
38
NYWXCSRDIEFVGLTMQP
A
H
3940
XIHCFWRST
L
V
G
P
K
Q
A
E
41
ARTXSLIVCN
Q
Y
D
H
K
E
42
LAVGQCEFDYPS
R
N
K
I
T
X
M
43
S
A
G
F
I
P
R
44
IXGTWEFDRSAVMP
L
H
Q
45
PWAGXEICSNMRTQ
K
H
L
F
Y
46
ARG
FWIXPVLSDECQ
47
IAMFDXPWTKQSER
V
H
L
Y
G
48
AXDRVKQEHPLGWTNS
M
F
C
I
49
PEDAXGLIYSTWRCFK
N
Q
50
XAIRTPDVWMGKQNCHL
E
Y
S
51
TLDQAPXIEKRSGVCNMHWF
52
GLIXAVEFWPMSTN
53
TPXSDLHCVRGFMK
54
REKTWSAIN
D
P
55 5657 58596
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
6
9
70 71727374
IARXKFTDNWGMSCLVEHQ
Y
75
LEQGWCXYFARTDKPVIHMN
76
XSVIAMFDK
N
T
R
G
Q
W
L
7778 79
RTWASHCKLQ
F
M
N
Y
X
V
I
8081
AGETQI
H
D
N
R
C
828384 85
PRKNDIYQWSLXAVTC
86
LGIPVTSARHYXWN
87
XPYSRLNQCEDKVTMWGA
H
F
88
G
DNLT
S
EWIX
RFYC
8990
XAENDWHSRVGKILTYCMF
91
SAFMCELVGTYIKP
X
N
Q
R
W
92
NHEQTGXIPRLMAFKVYWS
93
KSMWXFNILCVRPTAGDHE
9495
RYQMXCFAEHTIWSLK
G
V
P
N
96
XEFRIHVQAC
N
M
G
L
Y
T
P
S
97
KFYMALIWGRSXNTCPDH
98
PTLHXEGFNK
D
V
I
R
Y
S
A
99
PSVGTDRIHCFLAQWE
K
100
SVRGNITCAKFYQDXLHEMW
101
LRPAVDIQHX
M
S
K
NT
G
C
102
LPXFKYQWAHMCISNV
103
Y
104 105
LPXVARHGMIF
S
Y
0
1
2
3
4
bit
s
0
LTPVC
F
K
Y
Q
S
G
H
1
XPWNCGHSEIYAVLRTFKMD
2
GTRLNEKFVCYMIDSWQAXP
3
XMCETSDYLVKR
G
I
A
Q
P
H
W
4
IERKNVDFMWTPLHYCSQGXA
5
DIRFNQHATCSPLYWVGMX
6
PTIWRQVACNLEDK
M
S
F
X
Y
7
EVACSTQIWNMGFYDRPHXL
8
LRIVGFTSNY
9
IRAVGWTCY
Q
E
D
P
X
H
S
L
10
SYCMVKQGENTFLWRDAHIPX
1112
ATISVKWRMCGFE
Q
Y
D
P
H
L
X
13
LPAREKDYWIT
S
Q
14
AIPVFTDQWG
H
X
M
C
R
E
NY
S
L
15 16 17
GQRTNCEAXMSDPIVFKHY
18
VAG
P
S
C
R
E
D
L
H
K
T
I
19
LXRFYIQG
T
S
V
20 2122 23
RGIEKTY
Q
S
L
C
N
X
PV
A
24
TPAKGLDYWSHVRNI
25
AL
I
P
E
K
D
Q
G
T
M
YS
26
TPVKLQHXAIRESNDY
27
RF
T
Y
Q
I
S
V
C
G
N
PL
28
SYCQHDEGNRKTVXIWFPAM
29
AGTLVRKNIHMPDY
Q
S
E
C
30
XDWGQHLSFRANPVCYTIMK
31
RGLPEKFDNWAIVX
C
M
S
Y
32
FEGNHQVPTIWRLASYDMX
33
AGXPRELDQVIWHSNTCFKY
34
XKNYASHTEPWIFMRQLGC
35
VGMPDTAINRKYLSQEHFCWX
36 37
GTWEKFLYVADICRHQN
M
P
X
38
TIPRKDLGHXESWVQ
39 40
XEQMARLPVGTSI
C
H
N
Y
D
F
41
LREGYIXF
M
NS
A
P
T
42
SIVKFTNCDWGL
R
P
Q
H
43
ATIMKSG
Y
R
F
L
Q
H
P
44
PVRTNDYISAHXMELG
45
MCFWHPLKS
R
T
I
E
G
D
A
V
46
YWAHLNKCRSVIPG
T
D
M
E
47
LKDQWHVGFAEC
P
T
S
R
N
M
I
48 49
KFXNRQTSWHGMILVADEP
50
XQLENSHTMRIGDWACVKPF
51
HCQESLYWVGMKNAIDRX
P
T
52
RLPADYIWSTHM
5354
TXSFNYLIPREHC
55 56 575859 6
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
6
9
70 71 7273 74
EYCQSHDGLNMTVFPRKAIXW
75
GTISFNDRMCALYWQ
76
AVREKIYSHGTLNFC
77
CATQVRKNIFYGXWSLDMPH
78
DNGHCQXKLMTPSYWRIEAVF
79
NIVCAXTRFYQKPLMGWEHSD
80
MVFNCKYLWHTGIDPRXSEQA
81 82
ATLISKQXMRE
83
LARETWS
Y
F
G
Q
84
SXAVEKFYQWLTMRNI
8586
SEFT
D
Y
I
M
K
P
G
87
SPFHGAXRTQM
K
V
W
D
N
I
L
88
XVKFTQSL
I
G
P
A
89
GXAEFTDSIN
K
90
LSTDY
Q
A
I
R
G
M
N
91
REDQHPSGKNIYXL
V
T
A
F
C
92
ASYHIMVKRWTLQCEGPXND
93
GXEFWSHPMAILNTVYKCQ
94
VREFNQSAGXMPITLWK
95
YXKQLHPSGIFMTVDNEWACR
96
GLSPYVTWQHA
C
R
F
D
N
I
E
K
97
AXSGYWPTQF
L
K
N
D
V
I
R
E
98
SIPQWAHGVDYRKF
C
L
T
M
X
E
N
99
XPAYQSCDIWMKVE
F
H
N
R
L
G
T
100
TSRIYQA
L
H
X
M
W
E
KP
101
ATIXKGHFLE
R
S
Q
N
D
W
102 103
RLVAGYSHCEIQPTX
104 105
LTISVKNRGXEYWA
0
1
2
3
4
bit
s
0
EF
DYWS
R
LHTPANGC
1
IARTNYWLGV
C
S
FX
P
2
ISAQXYWVEK
C
F
H
N
R
L
T
G
3
AQSIGYDRKLTC
F
M
E
X
N
4
SWAHGQXNYPTFL
K
V
D
R
I
E
5
XLSYGVTPHWQFCARNIDE
K
6
XIGLHKMSQVPTDNAWCE
R
7
RGIXVENATSDPLFK
89
IAXPSVRFTLGNYCK
Q
E
10
IXATRLGHMC
E
D
V
K
S
Y
11
XEDWGLQSNRHTVYIPAKCFM
12
GLAVFNIHXPMKTWY
Q
C
E
S
13
RIXAKLDWHEGYSTV
14
EDHGP
W
X
M
T
R
V
A
I
N
F
K
151617 18
LGTCWNIKSYEDRMXVPAQ
19
GYMADIRHKSTLNWP
X
F
20
PEKTRIGSFDNQH
X
21
AVKFTSRLIXMPGY
22
IPXAFQWSREKLYC
23
IRXAFDWKNPHQGT
L
M
C
S
E
Y
24
AGPFLIWHTCRDV
S
E
Y
25
TIPSARYQMCGEK
26 2728
GSVLQTPCIFKNYRE
29
AVRIWTMCFSKGE
Q
Y
D
H
P
L
X
3031
Y
DMICRHWVENKXPAGTLQF
32
XARNWLITPSVEFQCGH
33
FKCQNXRGWTSHMDIVLE
P
A
3435
ALEKD
P
R
T
S
N
M
I
36
XFNLQAHKRMDPI
S
G
V
T
E
37
XFYCNHMLPKS
R
T
D
I
V
A
E
G
38
PAQTMLVGWEIFRCSDXNYK
39
APXSVRTNDHML
40
FDNCGYVHATSPEIRQLKW
M
X
41
LIQATPMVKYX
S
C
G
W
R
42
RLPKTWSIXMCD
G
A
E
V
43
SRFWAXG
L
D
I
Q
H
T
44
GTXRKNWISYLPFVCHQAM
E
D
45
ATDHGSVLKYECIRQN
M
P
X
4647
VGMTAIPNDRKLYSHQEFWCX
48
TXSAVEKNIPRDLMYGFC
49
RGPXKDQWHLASIVNTFC
Y
50
APXKFTMGNICWRE
S
H
Y
L
Q
51
LSVTQRGXAIEKYPW
52
SGVDLRHXAIYW
K
53
GTISREKAMVXWD
54
ASKDLYWRGXC
I
55 5657 58596
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
6
9
7071727374
IPVRFGNDYAEKSLCQH
75
GVFDYQARTSLWEKPX
76
QGRTDKHANCVIFESMXPY
77
VGFDLSRHMEXPITWKYCQ
78
SAWMYHKIRPTELVXGCND
79
SDQWHMPIGREXKNYLT
V
F
C
A
8081 8283
AGXREKTLHPMVISC
84 85
AVREKGTDLQWSIF
86
IRGXAEFSTCKVQ
87
SPXEDQWHMVAGFLIYCTR
88
SDIWRLPXGEQTK
N
H
M
A
V
899091
IQCFVNXAYTWRPKLGH
D
S
E
M
92
HXGVKMLSCQPTYR
E
I
F
A
93
ECGDATPVQRKNILFSXMWY
94
ECVATSWQINXFMGYHLRPD
95
ATNQWVGRPCLDKE
M
S
F
X
Y
96
KNDICTRHSFAQPLWYGVMX
97
DIREKNVFHLPMTCWQYSXG
A
98
ATXSFYDMCLR
K
V
I
Q
G
H
P
W
99
GLERKNFMDWACVISYPQX
100
XNWEMLYKHCRIADGTFVS
101
ALXETIQSRCGWFYH
102 103 104
ARPDNILHTX
K
M
105
0
1
2
3
4
bit
s
0
S
W
EFAVT
Q
YRMLPN
KIX
1
AKNLYWRGSVIXC
E
2
GKYASRPMEFVILD
C
3
XAREGFKLIPMTSY
456
GLS
F
T
D
I
R
M
A
78
GLXREFDIWHTSK
9
10
LREDYWAGCKST
N
11
STLIAENWRGPKV
12 13 14
IRXPKFGTQSEAVH
15
IX
V
L
W
T
S
AF
16 17
RIPEGNDYQSHMALW
18 19
FIMAPKLRTVWDNHGSQYEC
20
PXGSDWFHTVNKERQILAMYC
21
SAXVEFTDQGC
I
R
22
ILSRFKTDNYWXEVHG
23
RGPSKL
Y
A
X
I
V
24
DISRMLHVPWCGTN
X
K
F
A
Q
25
SYECQKGMXWTRPAILNFVDH
26 27
ATXSGDQLIRH
N
E
28
RXSKTNDLGY
H
29 30
R
H
SACGFDPW
31
SALXVKTDNHMPEI
32
VARELDYQTINWP
33
ARTXVELIPGNQC
34
RGPXNMEFKTLYA
C
35 36
ASGTVFLNRDXPH
37
PVENARLIXCFWD
38
SLXREKNYAMIPWFC
39
ARLVGKTNDIHCS
40 41
X
FIYLGPANDQW
42 43 4445 46
LPGKNQSIACRTHF
47
REFGKNQWSALPCTD
48
AXSVEQLHIKFTDGRC
4950
ALPVGTSMI
Y
R
X
K
51
TIXPVEFRMG
A
K
D
C
S
L
Y
52 53
T
54 55 56 57 58 596
0
6
1
6
2
6
3
6
4
6
5
6
6
6
7
6
8
6
9
70 7172 7374
RPXVFNIC
L
G
Y
Q
T
H
D
S
75
PAKFTDIRXY
W
Q
C
G
E
S
L
7677
SLPAGDWRMCIFNT
78
SNIAGMPLFYQTE
79
AGTPXDNIQSLHVRK
80
PSEFDWRXLKHMV
81 8283
XEKDL
I
G
S
A
W
M
84 85
LPXSFDYWAIGE
86 87
SEFKWRLTVAIGD
M
88 89
LTISEGYARCKQX
90
TLPEX
M
I
G
N
R
W
91
IATPXVFGNWSLEKD
92 93 94
LTEGKNQIMFSAXHW
95
PVGNLQRHXMITSAKD
96
IAKTDLRGPMSWEQ
97 98
SIEGDLARMKYXF
99
100
SAVKNIYRLGPCE
101 102 103 104 105
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
0123456789
10 12 22 26 27 2829 30 32 3334 35 3637 3839 41 42 43 4445 4748 4950 51 5253 54 56 57 59 662 70 74757611 55 58 60 6364 65 6667 68 69 71 727323 24 25 31 40 46
1
e1
1
e1
2
e1
3
e1
4
e1
5
e1
6
c1 c2 c4 e2
7
e2
6
e1
7
c3 c5 e2
5
e2
4
e2
3
e2
2
e2
1
1314 1516 1717
A
18 19 2120 20
A
20
B
A-stem A-stemT-stemT-stemC-stemC-stemD-stemD-stem
Adenine
Cytosine
Guanine
Uracil
gap
Fatemeh Hadi-Nezhad
Travis Lawrence
Lawrence et al. (2021). Bioinformatics 37(20):3654
tSFM is freely available to compute CIFs (including base-pair CIFs)
for tRNAs (and other RNA, protein, and DNA element families)
https://github.com/tlawrence3/tSFM
Travis Lawrence
Lawrence et al. (2021). Bioinformatics 37(20):3654
tSFM is freely available to compute CIFs (including base-pair CIFs)
for tRNAs (and other RNA, protein, and DNA element families)
https://github.com/tlawrence3/tSFM
Outline tRNA CIFs Predict Functional
Differences Between Human and
Trypanosome aaRSs Rare tRNA Features are
Functionally Informative
A B
A
B1
B2+3
C1
E
C3
F
G
scores Cyano-MLP
13
0.0
1.0
classification
probabilities0
1
2
3
4
bits
1
AA
SSMM
XX
JJ
FFNN
QQ
2
GG
JJ
CC
WW
YY
HH
3
GG
NN
VV
LL
CC
TT
EE
4
TT
JJ
EE
VV
AA
CC
RR
WW
HH
NN
MM
KK
YY
5
LL
GGKK
QQ
TT
JJ
FF
NN
AA
RR
YY
WW
II
6
QQ
VV
EE
MM
YY
JJ
KK
NN
HH
LL
RR
GG
AA
PP
DD
TT
7
SS
RR
GGNN
YY
KK
AA
VV
WW
JJ
II
89
GG
JJ
FF
10
SS
RRYY
11
RR
AA
NN
LL
TT
PP
QQ
YY
HHDD
EE
GG
WW
12
PP
JJSSWW
KK
VV
GG
MM
TT
NN
RR
AA
II
FF
DD
13
KK
YY
JJ
LL
CC
TT
AA
RR
QQ
MM
WW
GG
EE
14
SS
AA
YY
15
KK
QQJJ
16
PP
II
EE
KK
XX
HH
QQ
RR
SSNN
JJ
DD
WW
YY
VV
AA
TT
GG
FF
CC
MM
LL
17
SS
YY
HH
EE
QQ
NN
KK
TT
VV
RR
DD
FF
LL
WW
PP
AA
II
18
SS
TT
EEXX
PP
19 20 21
EE
JJ
YYLLDD
WW
KK
AA
TT
SS
MM
QQ
HH
XX
NN
FF
GG
PP
II
CC
VV
22
GG
PP
FFWW
NN
XX
CC
KK
EE
JJ
VV
SS
MM
HH
YY
DD
RR
II
23
PP
DD
NN
SS
RREE
24
YY
MM
JJ
LL
KK
25
KK
NN
FF
SS
QQYY
26
TTYY
KK
SS
27
MM
QQ
YYXX
28
SS
QQ
JJ
FF
EE
MM
VV
LL
AA
YY
TT
WW
KK
GG
RR
29
RR
LL
MM
NN
GG
WW
30
PP
VV
SS
LL
QQ
YY
JJ
FF
EE
WW
AA
MM
NNKK
TT
RR
CC
GG
XX
31
RRYY
WW
QQ
JJ
CC
EE
PP
TT
LL
DD
SS
GG
VV
FF
KK
NN
AA
HH
32
GGJJ
YY
DD
LL
MM
KK
SS
FF
TT
RR
AA
QQ
VV
PP
EE
33
AASS
34
VV
SS
JJ
EE
AA
MM
NN
TT
RR
DD
LL
GG
YY
XX
PP
FF
QQ
HH
CC
WW
35
KK
YYNN
FF
EE
MM
SS
RR
AA
GG
LL
QQ
VV
TT
HH
PP
36 37
SS
LL
TT
GG
PP
VV
RR
AA
KK
QQ
EE
38
NN
QQ
EE
HH
DD
YY
KK
39
SS
RR
KK
TT
XX
MM
II
JJ
NN
40AA
41
SSGG
RR
AA
PP
LL
VV
QQ
HH
42
NN
GG
QQ
EE
RR
AA
KK
HH
TT
YY
FF
LL
SS
JJ
43
TT
GG
LL
EE
DD
NN
AA
QQ
SS
MM
RR
YY
CC
JJ
44
FF
AA
NN
HH
JJ
KK
XX
MM
YY
RR
PP
VV
SS
LL
GG
45
KK
GG
PP
DD
MM
HH
RR
SS
EE
LL
YY
TT
FF
CC
WW
II
46
QQ
JJ
FF
MM
GG
YY
RR
WW
TT
CC
LL
SS
KK
NN
47
AAQQ
FF
DDTT
WW
RR
JJ
EE
GG
MM
HH
CC
LL
YY
KK
NN
SS
48
TT
KK
HH
SS
NN
AA
JJ
LL
CC
MM
PP
VV
GG
RR
FF
WW
49
RR
KK
FF
EE
SS
JJ
CC
HH
LL
QQ
YY
50
YY
JJ
SS
LL
EE
CC
GGQQMM
WW
AA
KK
RR
FF
TT
NN
VV
PP
XX
II
DD
HH
51
SS
PP
FF
CC
HH
JJ
VV
YY
TT
AA
NN
RR
MM
WW
EE
GG
QQ
KK
DD
LL
52
GG
II
KK
53
VV
JJ
AA
CC
MM
LL
KK
GG
QQ
RR
WW
YY
TT
SS
FF
PP
HH
NN
54
QQ
TT
MM
LL
PP
NN
JJ
VV
XX
YY
II
55
GG
NN
YY
56 57 58 59YY
60 61 62
AA
RRDD
EE
CC
XX
KK
LL
QQ
MM
YY
FF
NN
TT
GG
HH
JJ
63
DD
QQ
FF
TT
YY
AA
HH
NN
RR
MM
EE
JJ
LL
KK
VV
GG
II
XX
SS
CC
PP
WW
64
VVYYEE
65
RR
VV
SS
NN
AA
GG
YY
XX
DD
WW
FF
TT
LL
CC
66
AA
SS
YY
FF
MM
XX
TT
LL
JJ
VV
RR
KK
PP
WW
NN
QQ
67
GG
MM
KK
YY
WW
LL
QQ
SS
RR
68
SS
NNMM
QQ
FF
KK
LL
JJ
GG
PP
TT
EE
XX
VV
AA
HH
DD
69
RRSS
JJ
TT
MMLL
KK
DD
NN
WW
YY
VV
GG
XX
AA
EE
FF
CC
70
KK
SS
GG
WW
NN
LL
RR
JJ
DD
QQ
AA
MM
FF
VV
II
71
YY
FF
NN
KK
JJ
WW
QQ
AA
CC
TT
GG
RR
LL
VV
HH
PP
SS
72
SS
YY
TT
GG
AA
NN
XXKK
JJ
RR
PP
MM
HH
FF
DD
73
RRTT
NN
JJ
WW
YY
VV
AA
SS
74
YY
DD
NN
XX
WW
RR
75
TT
GGWW
NN
VV
RR
MM
LL
YY
XX
EE
76
SS
KK
QQ
JJ
MM
EE
YYGG
TT
CC 0
1
2
3
4
bits
1
RR
PP
SS
LL
KK
EE
FF
MM
JJ
XXNN
QQ
2
SS
PPJJ
II
MM
WW
CC
HH
3
SS
VV
EE
RR
FF
JJ
CC
II
MM
LL
TT
4
LL
SS
FF
WW
AA
II
QQ
VV
CC
RR
HH
JJNN
MM
KK
YY
5
PP
SS
QQ
DD
HH
GG
KK
RR
LL
NN
EE
AA
FFWW
II
6
SS
WW
CC
KK
NN
MM
HH
QQ
EE
JJ
VV
LL
RR
GG
PP
TT
AA
DD
7
LL
RR
TT
QQ
YY
SS
NN
MM
KK
VV
AA
WW
JJ
II
89
10
WWSSYY
11
VV
SS
LL
HH
PP
RR
QQEE
GG
DD
WW
12
EE
CC
PP
SS
JJ
WW
TT
MM
RR
KK
GG
VV
FF
AA
II
NN
DD
13
PP
AA
SSNN
DD
CC
KK
MM
JJ
YY
RR
QQ
WW
GG
EE
14 15
AA
SS
NN
YY
KK
MM
XX
JJ
16
II
PP
XX
EE
KK
JJ
QQ
WW
LL
MM
HH
RR
NN
YY
DD
VV
SS
TT
AA
GG
CC
FF
17
SS
TTNN
EE
KK
YY
HH
QQ
JJ
RR
MM
WW
VV
DD
FF
II
LL
PP
AA
18
TT
II
FF
JJ
YYMMPP
XX
19 20 21
EE
LL
JJ
QQ
NN
WW
MM
KK
AA
DD
SS
GG
TT
PP
CC
YY
VV
XX
FF
II
HH
22
LL
GG
KK
QQ
WW
AA
XX
CC
EEVV
SS
JJ
MM
RR
HH
YY
II
DD
23
LLPP
SS
DD
YY
RR
EE
24
SSRR
JJ
YY
KK
LL
25
QQFF
SSYY
26
VV
YYKK
TT
SS
27
JJMM
QQ
EEXX
28
PP
TTEE
YYCC
DD
AA
RR
LL
VV
QQ
SS
JJ
WW
MM
GG
KK
29
RR
SS
PP
VV
WW
KK
LL
GG
30
LL
NN
JJ
AA
FF
DD
MM
QQ
EE
KK
CC
SS
WW
RR
TT
GG
XX
31
MM
II
FFQQ
WW
RR
SS
TT
DD
CC
JJ
EE
PP
AA
GG
VV
LL
KK
HH
NN
32
WW
JJ
YY
HH
IIGG
DD
SS
LL
KK
QQ
TT
RR
FF
VV
PP
AA
EE
33
LL
GG
QQ
EE
SS
34
AA
MM
KK
SS
VVGG
XX
QQ
RR
DD
EE
LL
YY
FF
PP
HH
WW
CC
35
KK
II
NN
CC
MM
XX
WW
DD
EE
FF
SS
RR
VV
AA
GG
LL
TT
QQ
HH
PP
36 37
IISS
RR
LL
TT
PP
GG
VV
KK
AA
EE
QQ
38
YY
NN
QQ
DD
HH
KK
EE
39
SS
RR
MM
XX
TT
JJ
KK
NN
II
40DD
41
SS
EEGG
AA
RR
PP
VV
LL
QQ
HH
42
PP
XX
II
QQ
WW
EE
MM
AA
RR
HH
KK
NN
LL
TT
FF
JJ
YY
SS
43
II
KK
FF
CC
RR
HH
SS
QQ
MM
JJ
44
AA
CC
HH
WW
YY
MM
KK
JJ
XX
SS
PP
RR
VV
LL
GG
45
KK
NN
EE
RR
PP
QQ
JJ
VV
GG
LL
SS
HH
TT
MM
FF
WW
CC
II
46
PP
II
VV
QQ
EE
DDLL
TT
WW
GG
MM
CC
SS
JJ
RR
YY
KK
NN
47
TTMM
RR
HH
JJ
WW
GG
CC
EE
LL
NN
KK
YY
SS
48
IITT
QQ
YY
XX
DD
NN
CC
MM
LL
KK
RR
PP
JJ
SS
GG
VV
AA
WW
FF
49
PP
II
RR
KK
SS
MMJJ
LL
HH
CC
QQ
YY
50
SS
EE
YY
CC
LL
JJ
QQ
PP
GG
MM
KK
RR
WW
TT
HH
XX
DD
FF
NN
AA
II
VV
51
PP
CCHH
SS
VV
FF
AA
TT
MM
EE
YY
RR
JJ
QQ
NN
WW
GG
KK
DD
LL
52
LL
AASS
PP
53
EE
YY
AA
LL
XXCC
DD
SS
RR
JJ
WW
GG
VV
FF
KK
PP
NN
TT
MM
HH
QQ
54
SS
TT
FF
WW
LL
QQ
JJ
PP
NN
MM
VV
YY
XX
II
55
SSJJMM
56
LL
VV
WW
57 58 59
SS
QQ
RR
60 61 62
RR
DD
PP
SS
VV
AA
NN
QQ
YY
MM
EE
CC
KK
HH
LL
JJ
FF
GG
TT
63
DD
TT
RR
QQ
FF
KK
HH
AA
CC
YY
WW
SS
NN
PP
LL
GG
JJ
EE
VV
MM
XX
II
64
LL
YY
AAEE
65
PP
JJ
HHDD
XX
RR
GG
YY
NN
KK
EE
MM
SS
VV
AA
CC
LL
TT
FF
66
EE
FF
II
SS
XX
TT
MM
VV
YY
KK
LL
RR
PP
NN
JJ
QQ
WW
67
AA
PP
QQ
YY
MM
WW
XX
KK
LL
SS
RR
68
II
QQ
RR
SS
NN
MM
KK
JJ
EE
LL
TT
PP
GG
FF
XX
VV
AA
HH
DD
69
KK
QQDDJJHH
RR
LL
MM
SS
NN
TT
AA
WW
YY
VV
GG
EE
FF
CC
XX
70
PPHH
EEVV
CC
SS
QQ
JJ
AA
RR
LL
FF
KK
NN
MM
II
71
GG
EE
KK
NN
QQ
RR
WW
DD
JJ
AA
VV
TT
CC
PP
LL
HH
SS
72
LL
AA
SS
WW
GG
KK
NN
MM
JJ
TT
RR
PP
FF
DD
HH
73
TT
II
RR
KK
MM
NN
QQ
WW
VV
JJ
SS
AA
74
SS
PP
KK
VV
QQ
EE
WW
JJDD
XXRR
75
KK
SS
XX
NN
QQ
VV
WW
RR
JJ
MM
LLEE
76
RR
LLKK
FF
SS
XX
EETT
GG
CC 0
1
2
3
4
bits
1
AA
GG
VV
KKNN
QQ
23
LL
RR
VV
FF
EE
CC
TT
4
AA
GG
VV
LL
CC
DD
HH
TT
JJ
RR
WW
MM
KK
NN
YY
5
LLJJ
EE
NN
YY
KK
AA
TT
MM
DD
RR
WW
II
6
SSII
JJ
CC
MM
KK
EE
WW
HHLL
VV
RR
TT
PP
AA
QQ
GG
DD
7
SS
PP
LL
RR
NN
KK
QQMM
TT
AA
VV
JJ
WW
II
89
10
SSYY
11
SS
AA
PP
RR
HH
TTLLGG
DD
EE
WW
12
PPEEJJ
SSMM
VV
WW
TT
GG
NN
DD
KK
RR
AA
FF
II
13
SS
KK
JJ
DD
HH
TT
MM
QQ
WW
RR
GG
EE
14 15 16
PP
II
QQ
JJKK
EE
DD
VV
NN
RR
HH
SS
XX
MM
TT
CC
FF
GG
YY
WW
AA
LL
17
SSNN
QQ
CC
EE
YY
JJ
KK
RR
TT
VV
MM
LL
DD
FF
II
PP
WW
AA
18
FFJJXX
PP
19 20 21
R
EETT
KK
WW
LL
NN
QQ
VV
GG
PP
AA
II
SS
YY
JJ
DD
FF
XX
CC
HH
MM
22
TT
PP
GG
EE
CCVV
SS
JJ
MM
RR
HH
II
DD
YY
23
PP
DD
SSRREE
24
LL
MM
JJ
25
TT
PPHH
QQ
SSYY
26
PP
GG
TT
JJ
MMSS
27
AA
PP
KK
EE
JJXX
28
SS
LL
JJ
EE
NN
GG
PP
WW
RR
TT
KK
DD
AA
VV
29
RR
PP
EE
NN
GG
TT
VV
LL
30
PP
LL
II
EE
VV
FF
CC
SS
MMTT
DD
QQ
AA
JJ
KK
RR
WW
GG
XX
31
EE
XX
RR
JJ
IIYY
CC
FF
SS
VV
DD
TT
GG
AA
PP
LL
HH
KK
NN
32
LLGG
II
KK
NN
CC
YY
QQ
FF
SS
DD
RR
TT
AA
PP
VV
EE
33
LL
AA
EE
SS
34
GG
VV
II
RR
AA
JJ
EE
DD
FF
TT
LL
QQ
SS
HH
PP
CC
WW
35
II
KK
EE
MMFF
SS
CC
RR
AA
VV
GG
LL
TT
QQ
PP
HH
36 37
FF
IISS
RR
PP
TT
GG
LL
AA
VV
KK
EE
QQ
38
FFNN
EE
DD
QQ
KK
HH
YY
39
FFSS
RR
JJ
XX
KK
II
NN
MM
TT
40
RRDD
41
KK
GG
MMRR
AA
PP
LL
VV
QQ
HH
42
VV
GG
XXEE
QQ
CC
NN
MM
KK
AA
RR
HH
JJ
TT
SS
LL
FF
YY
43
TT
KK
LL
WW
SS
EE
YY
RR
MM
CC
QQ
JJ
44
TTJJ
YY
WW
HH
KK
CC
VV
MM
AA
SS
RR
PP
LL
GG
45
PP
KK
EE
MM
RR
GG
HH
LL
SS
VV
TT
WW
CC
FF
II
46
PPVV
QQII
YY
GG
LL
MM
TT
SS
RR
JJ
CC
KK
NN
47
II
VV
WW
HH
MM
RR
JJ
TT
GG
CC
LL
NN
KK
SS
YY
48
LL
NN
HH
TT
EE
DD
JJ
KK
SS
RR
PP
VV
AA
GG
FF
WW
49
AA
PPEE
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50
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51
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52
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53
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54
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55
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56 57 58 59 60 61 62
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63
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64
PPAAEE
65
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II
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RR
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AA
NN
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66
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67
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68
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69
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70
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71
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72
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73
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74
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RR
75
PP
VV
NN
LL
MM
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RR
WW
XXEE
76
RRKKGG
TT
CC 0
1
2
3
4
bits
1
AAMMNN
QQ
2
EEMM
3
WWRR
4
VV
EE
RRWWMM
KK
YY
NN
5
AAII
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RR
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6
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GG
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7
GG
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AA
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II
VV
89
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HH
GG
10 11
CCSS
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12
PPSS
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GG
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RR
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AA
FF
13
VVGG
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14
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15 16
MM
NN
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TT
VV
PP
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RR
AA
QQ
XX
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17
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18XX
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19 20 21
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22
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23
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24
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25
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26
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27
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28
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29SS
30
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31
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32
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33
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34
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35
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36 37
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38
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QQ
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39
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RR
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40 41
MM
VVRR
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AA
LL
PP
QQ
42
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RR
NN
CC
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YY
FF
43
LLQQ
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44
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45
LL
MM
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46
LL
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47
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48
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49
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50
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51
EEGGDD
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52
RRTT
53
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54
XX
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II
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55
RR
CC
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MM
56 57 58 59 60 61 62
RREETT
VV
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JJ
63
VV
DD
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HH
GG
QQ
RR
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FF
MM
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CC
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64 65
RR
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66
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QQ
67
LL
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68
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69
PPTT
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70
TTWW
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71
MM
AA
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RR
PP
GG
LL
SS
72
RRLL
PP
MM
HH
FF
DD
73
LLAA
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74
MMRR
75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
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NN
23
LL
EE
TT
4
RR
JJ
TTNN
WW
KK
MM
YY
5
LL
TT
FF
KK
II
6
NN
VV
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89
10 11
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13
RR
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14 15 16 17
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AA
18
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19 20 21
E
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22
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23
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24
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25
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26SS
27
QQ
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28
LL
QQ
DD
29 30
SS
QQ
RR
KK
JJ
WW
GG
XX
31
AA
VV
SS
YY
TT
GG
PP
LL
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NN
HH
32
LLGG
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RR
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VV
AA
33 34
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DD
KK
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PP
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35
NN
RR
VV
QQ
AA
GG
LL
CC
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PP
36 37
SSPP
GG
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EE
38
KK
HH
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QQ
39
SS
RR
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XX
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KK
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II
40 41
RR
AA
PP
LL
VV
QQ
HH
42
AA
EE
RR
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SS
CC
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43
RR
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44
VV
RR
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LL
GG
45
GG
LL
TT
VV
EE
SS
WW
FF
II
CC
46
LL
JJ
QQ
RR
SS
MM
KK
CC
NN
47
JJ
HH
EE
GG
KK
MM
CC
NN
LL
YY
SS
48
JJ
LL
PP
RR
VV
AA
GG
FF
WW
49
JJ
SS
EE
LL
CC
QQ
HH
YY
50 51
RR
TT
EE
NN
GG
QQ
DD
LL
52 53
RR
JJ
FF
GG
LL
HH
TT
PP
YY
NN
KK
QQ
54
PP
RR
JJ
VV
XX
II
YY
55 56 57 58 59 60 61 62
LLTT
JJ
GG
63 64EE
65
RR
AA
GG
DD
LL
TT
CC
66
RR
TT
LL
KK
PP
JJ
WW
QQ
NN
67
GG
JJ
LL
WW
RR
SS
XX
68
FF
RR
EE
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LL
MM
PP
GG
DD
VV
TT
HH
AA
69
LL
TT
SS
GG
VV
AA
EE
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CC
70
RR
SS
AA
JJ
FF
QQ
LL
VV
YY
MM
NN
II
71
AA
GG
RR
TT
LL
PP
HH
SS
72
SS
TT
RR
PP
FF
HH
73
RR
JJ
VV
SS
AA
74RR
75
RR
LL
WWEE
76
QQTT
CC
GG 0
1
2
3
4
bits
1
GG
TT
FF
JJ
MM
HH
QQ
NN
2
RRFF
HH
CC
3
GG
VV
QQ
LL
TT
EE
4
JJ
WW
NN
MM
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YY
5
VV
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EE
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6
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VV
MM
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AA
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7
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AA
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89
10
HH
11
RR
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12
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13
QQ
RR
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14 15 16
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17
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18
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19 20 21 22
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RR
MM
II
JJ
23
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24
RR
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YY
25
SS
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YY
26
KKSS
27
VV
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28
RR
GG
VV
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AA
29
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QQ
30
LL
TT
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XX
31
WW
GG
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RR
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AA
PP
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32
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34
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35
FF
RR
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LL
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36 37
SS
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38
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40 41
GG
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42
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GG
45
LL
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II
46
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WW
LL
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SS
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CC
NN
47
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RR
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VV
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CC
48
SS
LL
RR
PP
VV
AA
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49
SS
RR
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LL
CC
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QQ
YY
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50 51
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52
VVKK
53
SS
VV
RR
MM
WW
CC
NN
TT
PP
LL
QQ
GG
HH
KK
54
RR
NN
VV
XX
YY
II
55 56 57 58 59 60 61 62
LL
KK
RR
TT
HH
YY
NN
GG
FF
63 64
AAEE
65
SS
GG
NN
YY
LL
MM
TT
CC
FF
66
LLGG
FF
SS
VV
RR
MM
YY
TT
KK
PP
NN
JJ
WW
QQ
67
GG
VV
LL
QQ
SS
RR
XX
68
RR
LL
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MM
FF
GG
PP
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HH
AA
69
SS
WW
VV
GG
EE
AA
CC
FF
70
TT
KK
GG
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AA
QQ
RR
LL
DD
VV
MM
NN
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71
FF
JJ
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MM
RR
WW
CC
AA
VV
TT
LL
HH
PP
SS
72
GG
TTJJ
MM
RR
PP
HH
FF
DD
73
RR
MM
VV
JJ
SS
AA
74
WW
RR
75
RR
WW
LL
YYEE
76
VV
EE
WW
QQGG
TT
CC 0
1
2
3
4
bits
1
JJ
DDQQ
NN
2HH
3
GG
VV
EE
LL
MM
DD
JJ
TT
4
AA
FF
RR
DD
CC
HH
WW
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YY
5
AA
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II
6
VV
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EE
HH
CC
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RR
GG
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7
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AA
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II
89
10 11
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14 15 16
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17
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PP
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WW
AA
II
18
RRJJ
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XX
19 20
DD
RR
21 22
GG
KK
EE
CC
VV
SS
MM
RR
HH
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II
YY
23
SSRR
DD
CCEE
24
SS
LL
25
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26
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27
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28
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FF
AA
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29
LL
GG
30
LL
EE
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WW
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AA
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XX
31
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AA
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VV
HH
KK
NN
32
SS
YY
CC
LL
RR
FF
QQ
VV
AA
TT
PP
GG
EE
33
SS
LL
MM
34
GG
RR
QQ
YY
LL
SS
TT
EE
PP
FF
HH
CC
MM
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35
SS
RR
EE
AA
LL
GG
QQ
HH
VV
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PP
36 37
SS
RR
TT
GG
LL
VV
AA
KK
PP
QQ
EE
38
KK
YY
NN
EE
QQ
HH
DD
39
SS
RR
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TT
II
40 41
RR
AA
PP
VV
LL
QQ
HH
42
GG
RR
EE
HH
AA
TT
KK
NN
JJ
LL
FF
SS
YY
MM
43
AA
LL
HH
44
QQ
MM
JJ
AA
WW
RR
PP
SS
GG
CC
VV
LL
45
PP
EE
GG
LL
MM
YY
SS
JJ
FF
HH
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CC
II
46
EE
GG
VV
LL
TT
SS
RR
NN
JJ
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MM
47
VV
MM
HH
WW
QQ
GG
NN
CC
LL
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SS
KK
48
AA
LL
KK
HH
PP
VV
GG
RR
FF
WW
49
KK
MM
LL
CC
JJ
YY
HH
QQ
50
S
LL
CC
EE
JJ
AA
GG
QQ
MM
NN
KK
PP
TT
RR
FF
WW
HH
II
VV
XX
DD
51
SS
RR
TT
VV
AA
NN
HH
WW
EE
QQ
MM
GG
JJ
KK
DD
LL
52
EE
PP
53
LLAA
EE
GG
QQ
RR
YY
CC
TT
II
FF
JJ
NN
KK
MM
HH
PP
54
LL
PP
KK
RR
CC
NN
VV
JJ
XX
YY
II
55 56 57 58 59 60 61 62
SS
TT
LL
KK
RR
PP
NN
QQ
FF
MM
HH
GG
JJ
63 64
AAEE
65
SS
RR
KK
EE
NN
DD
JJ
AA
MM
TT
GG
LL
FF
CC
66
SS
VV
TT
JJ
RR
LL
NN
PP
WW
QQ
67
PP
WW
JJ
RR
VV
GG
LL
SS
QQ
68
SS
KK
RR
II
CC
LL
JJ
EE
FF
PP
VV
GG
AA
TT
HH
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XX
69
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RR
DD
WW
CC
TT
SS
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MM
EE
FF
AA
YY
70
WW
CC
AA
LL
RR
QQ
VV
FF
KK
NN
II
71
KK
QQ
AA
CC
VV
TT
RR
GG
LL
HH
PP
SS
72
EE
GG
RR
JJ
PP
DD
HH
FF
MM
73
PP
JJ
WW
VV
AA
SS
74
VVRR
75
RR
VV
LLMM
EE
76
SS
KKTT
GG
CC 0
1
2
3
4
bits
1NN
QQ
23
NNTT
CC
4
RRMM
NN
WW
KK
YY
5
KK
VV
RR
LL
EEII
6
VV
LLRR
DD
GG
PP
AA
TT
7
KK
AA
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II
WW
89
10
SS
YY
11
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WW
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12
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NN
13
RR
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14 15 16
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NN
MM
HH
QQ
AA
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YY
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TT
GG
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WW
17
RR
QQ
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18
RRJJPP
XX
19 20 21
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AA
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TT
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XX
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PP
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22
AA
VV
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23
SS
RR
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24
GG
LLJJ
25
RRSS
26
RR
GGSS
27
JJXX
28
GG
SS
DD
VV
29
RRLL
MM
GG
30
SS
RR
TT
QQ
JJ
HH
PP
KK
GG
WW
XX
31
DD
RR
TT
VV
GG
PP
LL
AA
KK
NN
32
LLKK
QQ
SS
RR
TT
GG
AA
PP
VV
EE
33
VV
SS
34
TT
LL
GG
PP
SS
CC
WW
35
SSRR
MM
LL
AA
TT
GG
VV
HH
QQ
PP
36 37
SS
RR
GG
TT
PP
VV
LL
AA
EE
QQ
KK
38
HH
YY
QQ
DD
KK
NN
EE
39
SS
RR
II
XX
MM
NN
TT
KK
JJ
40 41
VV
RR
LL
AA
PP
HH
QQ
42
RR
KK
SS
AA
NN
TT
LL
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FF
HH
YY
43
PP
RRJJ
44
RR
TT
SS
QQ
PP
LL
VV
JJ
GG
45
AA
GG
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SS
WW
LL
EE
CC
HH
FF
II
46
TT
GG
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CC
YY
LL
RR
SS
NN
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MM
47
RR
GG
HH
CC
MM
SS
LL
NN
KK
YY
48
KK
GG
VV
RR
JJ
FF
WW
49
RR
LL
KKJJ
QQ
HH
CC
YY
50
LL
KK
JJ
CCVV
RR
QQ
PP
GG
FF
DD
AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
QQ
JJ
GG
DD
LL
52 53
RR
KK
LL
QQ
NN
PP
TT
GG
SS
HH
YY
54
RR
NN
JJ
VV
QQ
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
RRTT
KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
QQ
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
QQ
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
QQ
FF
EE
70
RR
VV
QQ
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
genera
Oscillatoria
Arthrospira
Anabaena
Nostoc
Cyanothece
Spirulina
clades
Pleurocapsa
Prochlorococcus
Gloeobacter
Pseudanabaena
Acaryochloris
Nodosilinea
function logos
Genome
g
tRNA
genes
T
g
C
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g
Nonetheless, tRNA CIFs are Highly
Evolutionarily Conserved tRNA Class Informative Features
(CIFs) are Grounded in Physics,
not Evolutionary Conservation
Differences Between Human and
Trypanosome aaRSs Rare tRNA Features are
Functionally Informative
A B
A
B1
B2+3
C1
E
C3
F
G
scores Cyano-MLP
13
0.0
1.0
classification
probabilities0
1
2
3
4
bits
1
AA
SSMM
XX
JJ
FFNN
2
GG
JJ
CC
WW
YY
HH
3
GG
NN
VV
LL
CC
TT
EE
4
TT
JJ
EE
VV
AA
CC
RR
WW
HH
NN
MM
KK
YY
5
LL
GGKK
TT
JJ
FF
NN
AA
RR
YY
WW
II
6
VV
EE
MM
YY
JJ
KK
NN
HH
LL
RR
GG
AA
PP
DD
TT
7
SS
RR
GGNN
YY
KK
AA
VV
WW
JJ
II
89
GG
JJ
FF
10
SS
RRYY
11
RR
AA
NN
LL
TT
PP
YY
HHDD
EE
GG
WW
12
PP
JJSSWW
KK
VV
GG
MM
TT
NN
RR
AA
II
FF
DD
13
KK
YY
JJ
LL
CC
TT
AA
RR
MM
WW
GG
EE
14
SS
AA
YY
15
KK
QQJJ
16
PP
II
EE
KK
XX
HH
RR
SSNN
JJ
DD
WW
YY
VV
AA
TT
GG
FF
CC
MM
LL
17
SS
YY
HH
EE
NN
KK
TT
VV
RR
DD
FF
LL
WW
PP
AA
II
18
SS
TT
EEXX
PP
19 20 21
EE
JJ
YYLLDD
WW
KK
AA
TT
SS
MM
HH
XX
NN
FF
GG
PP
II
CC
VV
22
GG
PP
FFWW
NN
XX
CC
KK
EE
JJ
VV
SS
MM
HH
YY
DD
RR
II
23
PP
DD
NN
SS
RREE
24
YY
MM
JJ
LL
KK
25
KK
NN
FF
SS
QQYY
26
TTYY
KK
SS
27
MM
YYXX
28
SS
JJ
FF
EE
MM
VV
LL
AA
YY
TT
WW
KK
GG
RR
29
RR
LL
MM
NN
GG
WW
30
PP
VV
SS
LL
YY
JJ
FF
EE
WW
AA
MM
NNKK
TT
RR
CC
GG
XX
31
RRYY
WW
JJ
CC
EE
PP
TT
LL
DD
SS
GG
VV
FF
KK
NN
AA
HH
32
GGJJ
YY
DD
LL
MM
KK
SS
FF
TT
RR
AA
VV
PP
EE
33
AASS
34
VV
SS
JJ
EE
AA
MM
NN
TT
RR
DD
LL
GG
YY
XX
PP
FF
HH
CC
WW
35
KK
YYNN
FF
EE
MM
SS
RR
AA
GG
LL
VV
TT
HH
PP
36 37
SS
LL
TT
GG
PP
VV
RR
AA
KK
EE
38
NN
EE
HH
DD
YY
KK
39
SS
RR
KK
TT
XX
MM
II
JJ
NN
40AA
41
SSGG
RR
AA
PP
LL
VV
HH
42
NN
GG
EE
RR
AA
KK
HH
TT
YY
FF
LL
SS
JJ
43
TT
GG
LL
EE
DD
NN
AA
SS
MM
RR
YY
CC
JJ
44
FF
AA
NN
HH
JJ
KK
XX
MM
YY
RR
PP
VV
SS
LL
GG
45
KK
GG
PP
DD
MM
HH
RR
SS
EE
LL
YY
TT
FF
CC
WW
II
46
JJ
FF
MM
GG
YY
RR
WW
TT
CC
LL
SS
KK
NN
47
AAQQ
FF
DDTT
WW
RR
JJ
EE
GG
MM
HH
CC
LL
YY
KK
NN
SS
48
TT
KK
HH
SS
NN
AA
JJ
LL
CC
MM
PP
VV
GG
RR
FF
WW
49
RR
KK
FF
EE
SS
JJ
CC
HH
LL
YY
50
YY
JJ
SS
LL
EE
CC
GGQQMM
WW
AA
KK
RR
FF
TT
NN
VV
PP
XX
II
DD
HH
51
SS
PP
FF
CC
HH
JJ
VV
YY
TT
AA
NN
RR
MM
WW
EE
GG
KK
DD
LL
52
GG
II
KK
53
VV
JJ
AA
CC
MM
LL
KK
GG
RR
WW
YY
TT
SS
FF
PP
HH
NN
54
TT
MM
LL
PP
NN
JJ
VV
XX
YY
II
55
GG
NN
YY
56 57 58 59YY
60 61 62
AA
RRDD
EE
CC
XX
KK
LL
MM
YY
FF
NN
TT
GG
HH
JJ
63
DD
FF
TT
YY
AA
HH
NN
RR
MM
EE
JJ
LL
KK
VV
GG
II
XX
SS
CC
PP
WW
64
VVYYEE
65
RR
VV
SS
NN
AA
GG
YY
XX
DD
WW
FF
TT
LL
CC
66
AA
SS
YY
FF
MM
XX
TT
LL
JJ
VV
RR
KK
PP
WW
NN
67
GG
MM
KK
YY
WW
LL
SS
RR
68
SS
NNMM
FF
KK
LL
JJ
GG
PP
TT
EE
XX
VV
AA
HH
DD
69
RRSS
JJ
TT
MMLL
KK
DD
NN
WW
YY
VV
GG
XX
AA
EE
FF
CC
70
KK
SS
GG
WW
NN
LL
RR
JJ
DD
AA
MM
FF
VV
II
71
YY
FF
NN
KK
JJ
WW
AA
CC
TT
GG
RR
LL
VV
HH
PP
SS
72
SS
YY
TT
GG
AA
NN
XXKK
JJ
RR
PP
MM
HH
FF
DD
73
RRTT
NN
JJ
WW
YY
VV
AA
SS
74
YY
DD
NN
XX
WW
RR
75
TT
GGWW
NN
VV
RR
MM
LL
YY
XX
EE
76
SS
KK
JJ
MM
EE
YYGG
TT
CC 0
1
2
3
4
bits
1
RR
PP
SS
LL
KK
EE
FF
MM
JJ
XXNN
2
SS
PPJJ
II
MM
WW
CC
HH
3
SS
VV
EE
RR
FF
JJ
CC
II
MM
LL
TT
4
LL
SS
FF
WW
AA
II
VV
CC
RR
HH
JJNN
MM
KK
YY
5
PP
SS
DD
HH
GG
KK
RR
LL
NN
EE
AA
FFWW
II
6
SS
WW
CC
KK
NN
MM
HH
EE
JJ
VV
LL
RR
GG
PP
TT
AA
DD
7
LL
RR
TT
YY
SS
NN
MM
KK
VV
AA
WW
JJ
II
89
10
WWSSYY
11
VV
SS
LL
HH
PP
RR
QQEE
GG
DD
WW
12
EE
CC
PP
SS
JJ
WW
TT
MM
RR
KK
GG
VV
FF
AA
II
NN
DD
13
PP
AA
SSNN
DD
CC
KK
MM
JJ
YY
RR
WW
GG
EE
14 15
AA
SS
NN
YY
KK
MM
XX
JJ
16
II
PP
XX
EE
KK
JJ
WW
LL
MM
HH
RR
NN
YY
DD
VV
SS
TT
AA
GG
CC
FF
17
SS
TTNN
EE
KK
YY
HH
JJ
RR
MM
WW
VV
DD
FF
II
LL
PP
AA
18
TT
II
FF
JJ
YYMMPP
XX
19 20 21
EE
LL
JJ
NN
WW
MM
KK
AA
DD
SS
GG
TT
PP
CC
YY
VV
XX
FF
II
HH
22
LL
GG
KK
WW
AA
XX
CC
EEVV
SS
JJ
MM
RR
HH
YY
II
DD
23
LLPP
SS
DD
YY
RR
EE
24
SSRR
JJ
YY
KK
LL
25
QQFF
SSYY
26
VV
YYKK
TT
SS
27
JJMM
EEXX
28
PP
TTEE
YYCC
DD
AA
RR
LL
VV
SS
JJ
WW
MM
GG
KK
29
RR
SS
PP
VV
WW
KK
LL
GG
30
LL
NN
JJ
AA
FF
DD
MM
EE
KK
CC
SS
WW
RR
TT
GG
XX
31
MM
II
FFQQ
WW
RR
SS
TT
DD
CC
JJ
EE
PP
AA
GG
VV
LL
KK
HH
NN
32
WW
JJ
YY
HH
IIGG
DD
SS
LL
KK
TT
RR
FF
VV
PP
AA
EE
33
LL
GG
EE
SS
34
AA
MM
KK
SS
VVGG
XX
RR
DD
EE
LL
YY
FF
PP
HH
WW
CC
35
KK
II
NN
CC
MM
XX
WW
DD
EE
FF
SS
RR
VV
AA
GG
LL
TT
HH
PP
36 37
IISS
RR
LL
TT
PP
GG
VV
KK
AA
EE
38
YY
NN
DD
HH
KK
EE
39
SS
RR
MM
XX
TT
JJ
KK
NN
II
40DD
41
SS
EEGG
AA
RR
PP
VV
LL
HH
42
PP
XX
II
WW
EE
MM
AA
RR
HH
KK
NN
LL
TT
FF
JJ
YY
SS
43
II
KK
FF
CC
RR
HH
SS
MM
JJ
44
AA
CC
HH
WW
YY
MM
KK
JJ
XX
SS
PP
RR
VV
LL
GG
45
KK
NN
EE
RR
PP
JJ
VV
GG
LL
SS
HH
TT
MM
FF
WW
CC
II
46
PP
II
VV
EE
DDLL
TT
WW
GG
MM
CC
SS
JJ
RR
YY
KK
NN
47
TTMM
RR
HH
JJ
WW
GG
CC
EE
LL
NN
KK
YY
SS
48
IITT
YY
XX
DD
NN
CC
MM
LL
KK
RR
PP
JJ
SS
GG
VV
AA
WW
FF
49
PP
II
RR
KK
SS
MMJJ
LL
HH
CC
YY
50
SS
EE
YY
CC
LL
JJ
PP
GG
MM
KK
RR
WW
TT
HH
XX
DD
FF
NN
AA
II
VV
51
PP
CCHH
SS
VV
FF
AA
TT
MM
EE
YY
RR
JJ
NN
WW
GG
KK
DD
LL
52
LL
AASS
PP
53
EE
YY
AA
LL
XXCC
DD
SS
RR
JJ
WW
GG
VV
FF
KK
PP
NN
TT
MM
HH
54
SS
TT
FF
WW
LL
JJ
PP
NN
MM
VV
YY
XX
II
55
SSJJMM
56
LL
VV
WW
57 58 59
SS
RR
60 61 62
RR
DD
PP
SS
VV
AA
NN
YY
MM
EE
CC
KK
HH
LL
JJ
FF
GG
TT
63
DD
TT
RR
FF
KK
HH
AA
CC
YY
WW
SS
NN
PP
LL
GG
JJ
EE
VV
MM
XX
II
64
LL
YY
AAEE
65
PP
JJ
HHDD
XX
RR
GG
YY
NN
KK
EE
MM
SS
VV
AA
CC
LL
TT
FF
66
EE
FF
II
SS
XX
TT
MM
VV
YY
KK
LL
RR
PP
NN
JJ
WW
67
AA
PP
YY
MM
WW
XX
KK
LL
SS
RR
68
II
RR
SS
NN
MM
KK
JJ
EE
LL
TT
PP
GG
FF
XX
VV
AA
HH
DD
69
KK
QQDDJJHH
RR
LL
MM
SS
NN
TT
AA
WW
YY
VV
GG
EE
FF
CC
XX
70
PPHH
EEVV
CC
SS
JJ
AA
RR
LL
FF
KK
NN
MM
II
71
GG
EE
KK
NN
RR
WW
DD
JJ
AA
VV
TT
CC
PP
LL
HH
SS
72
LL
AA
SS
WW
GG
KK
NN
MM
JJ
TT
RR
PP
FF
DD
HH
73
TT
II
RR
KK
MM
NN
WW
VV
JJ
SS
AA
74
SS
PP
KK
VV
EE
WW
JJDD
XXRR
75
KK
SS
XX
NN
VV
WW
RR
JJ
MM
LLEE
76
RR
LLKK
FF
SS
XX
EETT
GG
CC 0
1
2
3
4
bits
1
AA
GG
VV
KKNN
23
LL
RR
VV
FF
EE
CC
TT
4
AA
GG
VV
LL
CC
DD
HH
TT
JJ
RR
WW
MM
KK
NN
YY
5
LLJJ
EE
NN
YY
KK
AA
TT
MM
DD
RR
WW
II
6
SSII
JJ
CC
MM
KK
EE
WW
HHLL
VV
RR
TT
PP
AA
GG
DD
7
SS
PP
LL
RR
NN
KK
QQMM
TT
AA
VV
JJ
WW
II
89
10
SSYY
11
SS
AA
PP
RR
HH
TTLLGG
DD
EE
WW
12
PPEEJJ
SSMM
VV
WW
TT
GG
NN
DD
KK
RR
AA
FF
II
13
SS
KK
JJ
DD
HH
TT
MM
WW
RR
GG
EE
14 15 16
PP
II
JJKK
EE
DD
VV
NN
RR
HH
SS
XX
MM
TT
CC
FF
GG
YY
WW
AA
LL
17
SSNN
CC
EE
YY
JJ
KK
RR
TT
VV
MM
LL
DD
FF
II
PP
WW
AA
18
FFJJXX
PP
19 20 21
R
EETT
KK
WW
LL
NN
VV
GG
PP
AA
II
SS
YY
JJ
DD
FF
XX
CC
HH
MM
22
TT
PP
GG
EE
CCVV
SS
JJ
MM
RR
HH
II
DD
YY
23
PP
DD
SSRREE
24
LL
MM
JJ
25
TT
PPHH
SSYY
26
PP
GG
TT
JJ
MMSS
27
AA
PP
KK
EE
JJXX
28
SS
LL
JJ
EE
NN
GG
PP
WW
RR
TT
KK
DD
AA
VV
29
RR
PP
EE
NN
GG
TT
VV
LL
30
PP
LL
II
EE
VV
FF
CC
SS
MMTT
DD
AA
JJ
KK
RR
WW
GG
XX
31
EE
XX
RR
JJ
IIYY
CC
FF
SS
VV
DD
TT
GG
AA
PP
LL
HH
KK
NN
32
LLGG
II
KK
NN
CC
YY
FF
SS
DD
RR
TT
AA
PP
VV
EE
33
LL
AA
EE
SS
34
GG
VV
II
RR
AA
JJ
EE
DD
FF
TT
LL
SS
HH
PP
CC
WW
35
II
KK
EE
MMFF
SS
CC
RR
AA
VV
GG
LL
TT
PP
HH
36 37
FF
IISS
RR
PP
TT
GG
LL
AA
VV
KK
EE
38
FFNN
EE
DD
KK
HH
YY
39
FFSS
RR
JJ
XX
KK
II
NN
MM
TT
40
RRDD
41
KK
GG
MMRR
AA
PP
LL
VV
HH
42
VV
GG
XXEE
CC
NN
MM
KK
AA
RR
HH
JJ
TT
SS
LL
FF
YY
43
TT
KK
LL
WW
SS
EE
YY
RR
MM
CC
JJ
44
TTJJ
YY
WW
HH
KK
CC
VV
MM
AA
SS
RR
PP
LL
GG
45
PP
KK
EE
MM
RR
GG
HH
LL
SS
VV
TT
WW
CC
FF
II
46
PPVV
QQII
YY
GG
LL
MM
TT
SS
RR
JJ
CC
KK
NN
47
II
VV
WW
HH
MM
RR
JJ
TT
GG
CC
LL
NN
KK
SS
YY
48
LL
NN
HH
TT
EE
DD
JJ
KK
SS
RR
PP
VV
AA
GG
FF
WW
49
AA
PPEE
SS
LL
MM
JJ
CC
HH
YY
50
SS
YY
EE
LL
JJ
CC
GGPP
MM
KK
VV
RR
TT
WW
AA
II
HH
FF
NN
DD
XX
51
AA
SS
JJ
II
MM
NN
YY
WW
HH
EE
TT
RR
KK
GG
DD
LL
52
RR
AA
PP
CC
LL
IIKK
53
AACC
MM
XX
II
FFWW
LL
JJVV
RR
YY
GG
SS
TT
PP
KK
HH
NN
54
SS
TT
GG
RR
LL
EE
NN
FFPP
JJVV
II
YY
XX
55
VV
MM
JJ
56 57 58 59 60 61 62
II
AA
LL
JJ
EE
NN
RR
HH
KK
TT
GG
FF
63
DD
TT
KK
VV
RR
HH
FF
EE
AA
NN
LL
YY
WW
JJ
PP
SS
XX
CC
GG
II
MM
64
PPAAEE
65
PP
II
DD
WW
SS
EE
VV
RR
JJ
GG
AA
NN
KK
FF
TT
LL
MM
CC
66
SSLL
XX
KK
II
VV
TT
MM
RR
PP
JJ
NN
WW
67
AA
GG
EE
VV
TT
XX
WW
YYKK
LL
RR
SS
68
KKSS
NN
II
MM
FFWW
LL
XX
RR
JJ
PP
GG
TT
VV
EE
AA
DD
HH
69
KK
DD
II
RR
WW
TT
NN
MM
LL
SS
JJ
GG
VV
AA
YY
CC
FF
XX
EE
70
SS
EE
DDWW
CCTT
GG
XX
YY
FF
JJ
RR
KK
LL
MM
AA
VV
NN
II
71
EE
NN
II
VV
KK
FF
GG
CC
TT
RR
AA
PP
LL
HH
SS
72
AA
GGSS
EE
NN
CC
XX
II
KK
MM
JJ
RR
TT
PP
FF
HH
DD
73
LL
PP
GGKK
EE
NN
WW
CC
TT
II
RR
MM
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74
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75
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EE
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CC 0
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GG
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49
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LL
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HH
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YY
50
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KK
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RR
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GG
FF
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AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
JJ
GG
DD
LL
52 53
RR
KK
LL
NN
PP
TT
GG
SS
HH
YY
54
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NN
JJ
VV
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
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KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
FF
EE
70
RR
VV
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
genera
Oscillatoria
Arthrospira
Anabaena
Nostoc
Cyanothece
Spirulina
clades
Pleurocapsa
Prochlorococcus
Gloeobacter
Pseudanabaena
Acaryochloris
Nodosilinea
function logos
Genome
g
tRNA
genes
T
g
C
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g
Nonetheless, tRNA CIFs are Highly
Evolutionarily Conserved tRNA Class Informative Features
(CIFs) are Grounded in Physics,
not Evolutionary Conservation
Vol.:(0123456789)1 3
Journal of Molecular Evolution (2021) 89:103–116
https://doi.org/10.1007/s00239-021-09995-z
ORIGINAL ARTICLE
Structural and!Genetic Determinants of!Convergence in!the!Drosophila
tRNA Structure–Function Map
Julie!Baker!Phillips
1,2
!· David!H.!Ardell
1,3
Received: 22 September 2020 / Accepted: 11 January 2021 / Published online: 2 February 2021
© The Author(s) 2021
Abstract
The evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional
conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve
Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at
single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at
the interface of the two major stacked helical domains. We applied our tRNA Structure–Function Mapper (tSFM) method
independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted
tRNA structure–function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within
the rapidly evolving ion-binding pocket—Cytosine 17 (C17), ancestrally informative for lysylation identity—independently
gained asparaginylation identity and substituted in parallel across tRNA
Asn
paralogs at least once, possibly multiple times,
during evolution of the genus. In D. melanogaster, most tRNA
Lys
and tRNA
Asn
genes are co-arrayed in one large heterologous
gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in
the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a
role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to
compensate for the convergent and parallel gains of C17 in tRNA
Asn
paralogs in that lineage. Our results underscore the
functional and evolutionary relevance of our tRNA structure–function map predictions and illuminate multiple genomic and
structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.
Keywords Class-informative feature (CIF)!· Ion-binding pocket!· Parallel substitutions!· Convergent evolution!· Structure–
function map
Introduction
Transfer RNAs (tRNAs) were the first family of RNAs to be
directly sequenced (Holley 1965) and the first to be solved
by X-ray crystallography (Holley et!al. 1965). Historically,
algorithms to estimate phylogeny and substitution patterns
were tested on tRNA genes (Cedergren et!al. 1981; Eigen
et!al. 1989). Early in the genome era, it was reported that
tRNA genes can evolve to switch their functional (codon-
reading) identities through anticodon shift substitutions,
which entail both synonymous and non-synonymous substi-
tutions in anticodons (Saks et!al. 1998). However, the small
sizes and high similarities of tRNA genes pose obstacles to
inferring their orthology, which is needed to better under-
stand the evolutionary processes underlying functional turn-
over of tRNA genes. An important step forward came from
the “micro-syntenic" approach to infer tRNA gene orthol-
ogy using flanking sequences, first applied in Drosophila
(Rogers et!al. 2010) and later to other eukaryotes (Rogers
and Gri"ths-Jones 2014). These studies revealed that func-
tional turnover of tRNA genes through anticodon shift sub-
stitutions is more frequent and widespread than previously
known. However, Rogers and Gri"ths-Jones (2014) were
Handling editor: David Liberles.
* David H. Ardell
[email protected]
1
Quantitative and!Systems Biology Program, University
of!California, Merced, CA!95343, USA
2
Department of!Biology, Cumberland University, 1
Cumberland Square, Lebanon, TN!37087, USA
3
Department of!Molecular and!Cell Biology, University
of!California, Merced, CA!95343, USA
Julie Phillips, Ph.D.
Phillips and Ardell (2021). J. Mol. Evol. 89:103
Journal of Molecular Evolution (2021) 89:103–116
https://doi.org/10.1007/s00239-021-09995-z
ORIGINAL ARTICLE
Structural and!Genetic Determinants of!Convergence in!the!Drosophila
tRNA Structure–Function Map
Julie!Baker!Phillips
1,2
!· David!H.!Ardell
1,3
Received: 22 September 2020 / Accepted: 11 January 2021 / Published online: 2 February 2021
© The Author(s) 2021
Abstract
The evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional
conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve
Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at
single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at
the interface of the two major stacked helical domains. We applied our tRNA Structure–Function Mapper (tSFM) method
independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted
tRNA structure–function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within
the rapidly evolving ion-binding pocket—Cytosine 17 (C17), ancestrally informative for lysylation identity—independently
gained asparaginylation identity and substituted in parallel across tRNA
Asn
paralogs at least once, possibly multiple times,
during evolution of the genus. In D. melanogaster, most tRNA
Lys
and tRNA
Asn
genes are co-arrayed in one large heterologous
gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in
the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a
role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to
compensate for the convergent and parallel gains of C17 in tRNA
Asn
paralogs in that lineage. Our results underscore the
functional and evolutionary relevance of our tRNA structure–function map predictions and illuminate multiple genomic and
structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.
Keywords Class-informative feature (CIF)!· Ion-binding pocket!· Parallel substitutions!· Convergent evolution!· Structure–
function map
Introduction
Transfer RNAs (tRNAs) were the first family of RNAs to be
directly sequenced (Holley 1965) and the first to be solved
by X-ray crystallography (Holley et!al. 1965). Historically,
algorithms to estimate phylogeny and substitution patterns
were tested on tRNA genes (Cedergren et!al. 1981; Eigen
et!al. 1989). Early in the genome era, it was reported that
tRNA genes can evolve to switch their functional (codon-
reading) identities through anticodon shift substitutions,
which entail both synonymous and non-synonymous substi-
tutions in anticodons (Saks et!al. 1998). However, the small
sizes and high similarities of tRNA genes pose obstacles to
inferring their orthology, which is needed to better under-
stand the evolutionary processes underlying functional turn-
over of tRNA genes. An important step forward came from
the “micro-syntenic" approach to infer tRNA gene orthol-
ogy using flanking sequences, first applied in Drosophila
(Rogers et!al. 2010) and later to other eukaryotes (Rogers
and Gri"ths-Jones 2014). These studies revealed that func-
tional turnover of tRNA genes through anticodon shift sub-
stitutions is more frequent and widespread than previously
known. However, Rogers and Gri"ths-Jones (2014) were
Handling editor: David Liberles.
* David H. Ardell
[email protected]
1
Quantitative and!Systems Biology Program, University
of!California, Merced, CA!95343, USA
2
Department of!Biology, Cumberland University, 1
Cumberland Square, Lebanon, TN!37087, USA
3
Department of!Molecular and!Cell Biology, University
of!California, Merced, CA!95343, USA
Julie Phillips, Ph.D.
Phillips and Ardell (2021). J. Mol. Evol. 89:103
Even though Drosophila tRNA genes do evolve …
107Journal of Molecular Evolution (2021) 89:103–116
1 3
aggregating over di!erent functional classes of tRNA gene
orthologs and using the fixed known species tree"(Drosoph-
ila 12 Genomes Consortium 2007). We fitted unpaired sites
and loops with the General Time Reversible model (Lanave
et"al. 1984; Tavaré 1986; Rodrıǵuez et"al. 1990) with invari-
ant sites (GTR + I), and paired-sites and stems with the
Doublet(GTR) + I"model, also with invariant sites (Ron-
quist et"al. 2012). We obtained very similar results fitting the
(GTR + I + Gamma) model (data provided in Supplementary
Materials) or using the Hasegawa–Kishino–Yano (HKY)
model"(Hasegawa et"al. 1985) with or without Gamma-dis-
tributed site-rate variation (Phillips 2003 and Figures"S1 and
S2 in Supplementary Online Materials). We ran MrBayes
with the option “ratemult = scaled,” which implies that all
rates reported and shown in Figs."1 and 2 are scaled so that
the mean rate across all site-partitions is 1.0 substitutions
per site or site-pair.
In our coarse-grained analysis of substitution rates by
secondary structural elements, we observed that, regardless
of which alignments we used, the largest substitution rates
occur in the D-loop and acceptor stem followed closely by
the T-loop"(Fig."1). Considering the relative constraint that
acceptor stems are expected to have from identity-driven
interactions with proteins"(Giegé et"al. 1998), the acceptor
stem shows a surprisingly high rate of evolution. Further-
more, regardless of which alignments or models we used,
in our analysis of substitution rates by individual sites, we
found that three sites—corresponding to Sprinzl coordinates
16, 17, and 60—showed markedly higher rates of substitu-
tion than any other, followed by sites 15, 45, and 59 (Fig."2).
Sprinzl coordinates 16, 17, and 60 are three among
eight sites that form an extended ion-binding pocket
(Sprinzl coordinates 15, 16, 17, 18, 19, 20, 59, and 60)
between the D- and T-arms at the interface of the two
tRNA helical domains"(Behlen et"al. 1990), as shown in
Fig."3. This pocket was first described as site 1 in the
original orthorhombic crystal form"(Holbrook et"al. 1977)
and site 3 in the monoclinic structure"(Jack et"al. 1977)
and was later shown to potentially bind different metal
ions including magnesium, cobalt, manganese, and
lead"(Jack et"al. 1977; Holbrook et"al. 1977; Behlen et"al.
1990; Shi and Moore 2000). More recent structural work
better resolved the structure of Sprinzl site 16, which is a
fairly conserved uracil among eukaryotes (Marck and
Grosjean 2002), most likely post-translationally modified
to dihydrouridine, and demonstrated the conformational
sensitivity of this to ionic conditions"(Shi and Moore
2000). The hypergeometric probability that three sites
with elevated substitution rates occur within the eight
110011100111
111111110111
0
1
2
3
4
50
1
2
3
4
5
0
1
2
3
4 110011100111
Fig. 1 95% Credible intervals, interquartile ranges, and medians of
relative substitution rates by secondary structural elements in Dros-
ophila tRNA genes as calculated in MrBayes 3.2.1 using the GTR + I/
Doublet(GTR + I) models for loops/stems and the partitioned concat-
enated alignments indicated by bit-sets as defined in Table"1
A Stem DStemD LoopDStemC Stem C StemC Loop A StemT Stem T StemT Loop
18 1014 2227 3932 444954 616673
16
0
2
4
6
8
10
17
60
0
2
4
6
8
16
17
60
17
60
0
2
4
6
8
16
111111110111
110011100111
101111110000
Fig. 2 95% Credible intervals, interquartile ranges, and medians
of relative substitution rates by individual site/Sprinzl coordinate in
Drosophila tRNA genes as calculated in MrBayes 3.2.1 using the
GTR + I model for individual sites and the partitioned concatenated
alignments indicated by bit-sets as defined in Table"1
107Journal of Molecular Evolution (2021) 89:103–116
1 3
aggregating over di!erent functional classes of tRNA gene
orthologs and using the fixed known species tree"(Drosoph-
ila 12 Genomes Consortium 2007). We fitted unpaired sites
and loops with the General Time Reversible model (Lanave
et"al. 1984; Tavaré 1986; Rodrıǵuez et"al. 1990) with invari-
ant sites (GTR + I), and paired-sites and stems with the
Doublet(GTR) + I"model, also with invariant sites (Ron-
quist et"al. 2012). We obtained very similar results fitting the
(GTR + I + Gamma) model (data provided in Supplementary
Materials) or using the Hasegawa–Kishino–Yano (HKY)
model"(Hasegawa et"al. 1985) with or without Gamma-dis-
tributed site-rate variation (Phillips 2003 and Figures"S1 and
S2 in Supplementary Online Materials). We ran MrBayes
with the option “ratemult = scaled,” which implies that all
rates reported and shown in Figs."1 and 2 are scaled so that
the mean rate across all site-partitions is 1.0 substitutions
per site or site-pair.
In our coarse-grained analysis of substitution rates by
secondary structural elements, we observed that, regardless
of which alignments we used, the largest substitution rates
occur in the D-loop and acceptor stem followed closely by
the T-loop"(Fig."1). Considering the relative constraint that
acceptor stems are expected to have from identity-driven
interactions with proteins"(Giegé et"al. 1998), the acceptor
stem shows a surprisingly high rate of evolution. Further-
more, regardless of which alignments or models we used,
in our analysis of substitution rates by individual sites, we
found that three sites—corresponding to Sprinzl coordinates
16, 17, and 60—showed markedly higher rates of substitu-
tion than any other, followed by sites 15, 45, and 59 (Fig."2).
Sprinzl coordinates 16, 17, and 60 are three among
eight sites that form an extended ion-binding pocket
(Sprinzl coordinates 15, 16, 17, 18, 19, 20, 59, and 60)
between the D- and T-arms at the interface of the two
tRNA helical domains"(Behlen et"al. 1990), as shown in
Fig."3. This pocket was first described as site 1 in the
original orthorhombic crystal form"(Holbrook et"al. 1977)
and site 3 in the monoclinic structure"(Jack et"al. 1977)
and was later shown to potentially bind different metal
ions including magnesium, cobalt, manganese, and
lead"(Jack et"al. 1977; Holbrook et"al. 1977; Behlen et"al.
1990; Shi and Moore 2000). More recent structural work
better resolved the structure of Sprinzl site 16, which is a
fairly conserved uracil among eukaryotes (Marck and
Grosjean 2002), most likely post-translationally modified
to dihydrouridine, and demonstrated the conformational
sensitivity of this to ionic conditions"(Shi and Moore
2000). The hypergeometric probability that three sites
with elevated substitution rates occur within the eight
110011100111
111111110111
0
1
2
3
4
50
1
2
3
4
5
0
1
2
3
4 110011100111
Fig. 1 95% Credible intervals, interquartile ranges, and medians of
relative substitution rates by secondary structural elements in Dros-
ophila tRNA genes as calculated in MrBayes 3.2.1 using the GTR + I/
Doublet(GTR + I) models for loops/stems and the partitioned concat-
enated alignments indicated by bit-sets as defined in Table"1
A Stem DStemD LoopDStemC Stem C StemC Loop A StemT Stem T StemT Loop
18 1014 2227 3932 444954 616673
16
0
2
4
6
8
10
17
60
0
2
4
6
8
16
17
60
17
60
0
2
4
6
8
16
111111110111
110011100111
101111110000
Fig. 2 95% Credible intervals, interquartile ranges, and medians
of relative substitution rates by individual site/Sprinzl coordinate in
Drosophila tRNA genes as calculated in MrBayes 3.2.1 using the
GTR + I model for individual sites and the partitioned concatenated
alignments indicated by bit-sets as defined in Table"1
Phillips and Ardell (2021). J. Mol. Evol. 89:103
107Journal of Molecular Evolution (2021) 89:103–116
1 3
aggregating over di!erent functional classes of tRNA gene
orthologs and using the fixed known species tree"(Drosoph-
ila 12 Genomes Consortium 2007). We fitted unpaired sites
and loops with the General Time Reversible model (Lanave
et"al. 1984; Tavaré 1986; Rodrıǵuez et"al. 1990) with invari-
ant sites (GTR + I), and paired-sites and stems with the
Doublet(GTR) + I"model, also with invariant sites (Ron-
quist et"al. 2012). We obtained very similar results fitting the
(GTR + I + Gamma) model (data provided in Supplementary
Materials) or using the Hasegawa–Kishino–Yano (HKY)
model"(Hasegawa et"al. 1985) with or without Gamma-dis-
tributed site-rate variation (Phillips 2003 and Figures"S1 and
S2 in Supplementary Online Materials). We ran MrBayes
with the option “ratemult = scaled,” which implies that all
rates reported and shown in Figs."1 and 2 are scaled so that
the mean rate across all site-partitions is 1.0 substitutions
per site or site-pair.
In our coarse-grained analysis of substitution rates by
secondary structural elements, we observed that, regardless
of which alignments we used, the largest substitution rates
occur in the D-loop and acceptor stem followed closely by
the T-loop"(Fig."1). Considering the relative constraint that
acceptor stems are expected to have from identity-driven
interactions with proteins"(Giegé et"al. 1998), the acceptor
stem shows a surprisingly high rate of evolution. Further-
more, regardless of which alignments or models we used,
in our analysis of substitution rates by individual sites, we
found that three sites—corresponding to Sprinzl coordinates
16, 17, and 60—showed markedly higher rates of substitu-
tion than any other, followed by sites 15, 45, and 59 (Fig."2).
Sprinzl coordinates 16, 17, and 60 are three among
eight sites that form an extended ion-binding pocket
(Sprinzl coordinates 15, 16, 17, 18, 19, 20, 59, and 60)
between the D- and T-arms at the interface of the two
tRNA helical domains"(Behlen et"al. 1990), as shown in
Fig."3. This pocket was first described as site 1 in the
original orthorhombic crystal form"(Holbrook et"al. 1977)
and site 3 in the monoclinic structure"(Jack et"al. 1977)
and was later shown to potentially bind different metal
ions including magnesium, cobalt, manganese, and
lead"(Jack et"al. 1977; Holbrook et"al. 1977; Behlen et"al.
1990; Shi and Moore 2000). More recent structural work
better resolved the structure of Sprinzl site 16, which is a
fairly conserved uracil among eukaryotes (Marck and
Grosjean 2002), most likely post-translationally modified
to dihydrouridine, and demonstrated the conformational
sensitivity of this to ionic conditions"(Shi and Moore
2000). The hypergeometric probability that three sites
with elevated substitution rates occur within the eight
110011100111
111111110111
0
1
2
3
4
50
1
2
3
4
5
0
1
2
3
4 110011100111
Fig. 1 95% Credible intervals, interquartile ranges, and medians of
relative substitution rates by secondary structural elements in Dros-
ophila tRNA genes as calculated in MrBayes 3.2.1 using the GTR + I/
Doublet(GTR + I) models for loops/stems and the partitioned concat-
enated alignments indicated by bit-sets as defined in Table"1
A Stem DStemD LoopDStemC Stem C StemC Loop A StemT Stem T StemT Loop
18 1014 2227 3932 444954 616673
16
0
2
4
6
8
10
17
60
0
2
4
6
8
16
17
60
17
60
0
2
4
6
8
16
111111110111
110011100111
101111110000
Fig. 2 95% Credible intervals, interquartile ranges, and medians
of relative substitution rates by individual site/Sprinzl coordinate in
Drosophila tRNA genes as calculated in MrBayes 3.2.1 using the
GTR + I model for individual sites and the partitioned concatenated
alignments indicated by bit-sets as defined in Table"1
107Journal of Molecular Evolution (2021) 89:103–116
1 3
aggregating over di!erent functional classes of tRNA gene
orthologs and using the fixed known species tree"(Drosoph-
ila 12 Genomes Consortium 2007). We fitted unpaired sites
and loops with the General Time Reversible model (Lanave
et"al. 1984; Tavaré 1986; Rodrıǵuez et"al. 1990) with invari-
ant sites (GTR + I), and paired-sites and stems with the
Doublet(GTR) + I"model, also with invariant sites (Ron-
quist et"al. 2012). We obtained very similar results fitting the
(GTR + I + Gamma) model (data provided in Supplementary
Materials) or using the Hasegawa–Kishino–Yano (HKY)
model"(Hasegawa et"al. 1985) with or without Gamma-dis-
tributed site-rate variation (Phillips 2003 and Figures"S1 and
S2 in Supplementary Online Materials). We ran MrBayes
with the option “ratemult = scaled,” which implies that all
rates reported and shown in Figs."1 and 2 are scaled so that
the mean rate across all site-partitions is 1.0 substitutions
per site or site-pair.
In our coarse-grained analysis of substitution rates by
secondary structural elements, we observed that, regardless
of which alignments we used, the largest substitution rates
occur in the D-loop and acceptor stem followed closely by
the T-loop"(Fig."1). Considering the relative constraint that
acceptor stems are expected to have from identity-driven
interactions with proteins"(Giegé et"al. 1998), the acceptor
stem shows a surprisingly high rate of evolution. Further-
more, regardless of which alignments or models we used,
in our analysis of substitution rates by individual sites, we
found that three sites—corresponding to Sprinzl coordinates
16, 17, and 60—showed markedly higher rates of substitu-
tion than any other, followed by sites 15, 45, and 59 (Fig."2).
Sprinzl coordinates 16, 17, and 60 are three among
eight sites that form an extended ion-binding pocket
(Sprinzl coordinates 15, 16, 17, 18, 19, 20, 59, and 60)
between the D- and T-arms at the interface of the two
tRNA helical domains"(Behlen et"al. 1990), as shown in
Fig."3. This pocket was first described as site 1 in the
original orthorhombic crystal form"(Holbrook et"al. 1977)
and site 3 in the monoclinic structure"(Jack et"al. 1977)
and was later shown to potentially bind different metal
ions including magnesium, cobalt, manganese, and
lead"(Jack et"al. 1977; Holbrook et"al. 1977; Behlen et"al.
1990; Shi and Moore 2000). More recent structural work
better resolved the structure of Sprinzl site 16, which is a
fairly conserved uracil among eukaryotes (Marck and
Grosjean 2002), most likely post-translationally modified
to dihydrouridine, and demonstrated the conformational
sensitivity of this to ionic conditions"(Shi and Moore
2000). The hypergeometric probability that three sites
with elevated substitution rates occur within the eight
110011100111
111111110111
0
1
2
3
4
50
1
2
3
4
5
0
1
2
3
4 110011100111
Fig. 1 95% Credible intervals, interquartile ranges, and medians of
relative substitution rates by secondary structural elements in Dros-
ophila tRNA genes as calculated in MrBayes 3.2.1 using the GTR + I/
Doublet(GTR + I) models for loops/stems and the partitioned concat-
enated alignments indicated by bit-sets as defined in Table"1
A Stem DStemD LoopDStemC Stem C StemC Loop A StemT Stem T StemT Loop
18 1014 2227 3932 444954 616673
16
0
2
4
6
8
10
17
60
0
2
4
6
8
16
17
60
17
60
0
2
4
6
8
16
111111110111
110011100111
101111110000
Fig. 2 95% Credible intervals, interquartile ranges, and medians
of relative substitution rates by individual site/Sprinzl coordinate in
Drosophila tRNA genes as calculated in MrBayes 3.2.1 using the
GTR + I model for individual sites and the partitioned concatenated
alignments indicated by bit-sets as defined in Table"1
Phillips and Ardell (2021). J. Mol. Evol. 89:103
… their tRNA Class-Informative Features are almost
completely invariant over 70 million years of evolution
Phillips and Ardell (2021). J. Mol. Evol. 89:103
111Journal of Molecular Evolution (2021) 89:103–116
1 3
Fig. 5 Conservation of tRNA single-site CIFs in Drosophila with
Musca domestica as an outgroup showing extensive conservation
of tRNA CIFs in Drosophila. Tree topology from Drosophila 12
Genomes Consortium (2007), with divergence dates from Tamura
et!al. (2003) and Hennig et!al. (1981)
completely invariant over 70 million years of evolution
Phillips and Ardell (2021). J. Mol. Evol. 89:103
111Journal of Molecular Evolution (2021) 89:103–116
1 3
Fig. 5 Conservation of tRNA single-site CIFs in Drosophila with
Musca domestica as an outgroup showing extensive conservation
of tRNA CIFs in Drosophila. Tree topology from Drosophila 12
Genomes Consortium (2007), with divergence dates from Tamura
et!al. (2003) and Hennig et!al. (1981)
tRNA Class-Informative Features are highly conserved
over 250 million years of trypanosome evolution
Adenine Function Logos in Leishmania and Trypanosoma
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
over 250 million years of trypanosome evolution
Adenine Function Logos in Leishmania and Trypanosoma
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
tRNA Class-Informative Features are conserved over billions of
years of divergence between plastids and Cyanobacteria
A B
A
B1
B2+3
C1
E
C3
F
G
scores Cyano-MLP
13
0.0
1.0
classification
probabilities0
1
2
3
4
bits
1
AA
SSMM
XX
JJ
FFNN
QQ
2
GG
JJ
CC
WW
YY
HH
3
GG
NN
VV
LL
CC
TT
EE
4
TT
JJ
EE
VV
AA
CC
RR
WW
HH
NN
MM
KK
YY
5
LL
GGKK
QQ
TT
JJ
FF
NN
AA
RR
YY
WW
II
6
QQ
VV
EE
MM
YY
JJ
KK
NN
HH
LL
RR
GG
AA
PP
DD
TT
7
SS
RR
GGNN
YY
KK
AA
VV
WW
JJ
II
89
GG
JJ
FF
10
SS
RRYY
11
RR
AA
NN
LL
TT
PP
QQ
YY
HHDD
EE
GG
WW
12
PP
JJSSWW
KK
VV
GG
MM
TT
NN
RR
AA
II
FF
DD
13
KK
YY
JJ
LL
CC
TT
AA
RR
QQ
MM
WW
GG
EE
14
SS
AA
YY
15
KK
QQJJ
16
PP
II
EE
KK
XX
HH
QQ
RR
SSNN
JJ
DD
WW
YY
VV
AA
TT
GG
FF
CC
MM
LL
17
SS
YY
HH
EE
QQ
NN
KK
TT
VV
RR
DD
FF
LL
WW
PP
AA
II
18
SS
TT
EEXX
PP
19 20 21
EE
JJ
YYLLDD
WW
KK
AA
TT
SS
MM
QQ
HH
XX
NN
FF
GG
PP
II
CC
VV
22
GG
PP
FFWW
NN
XX
CC
KK
EE
JJ
VV
SS
MM
HH
YY
DD
RR
II
23
PP
DD
NN
SS
RREE
24
YY
MM
JJ
LL
KK
25
KK
NN
FF
SS
QQYY
26
TTYY
KK
SS
27
MM
QQ
YYXX
28
SS
QQ
JJ
FF
EE
MM
VV
LL
AA
YY
TT
WW
KK
GG
RR
29
RR
LL
MM
NN
GG
WW
30
PP
VV
SS
LL
QQ
YY
JJ
FF
EE
WW
AA
MM
NNKK
TT
RR
CC
GG
XX
31
RRYY
WW
QQ
JJ
CC
EE
PP
TT
LL
DD
SS
GG
VV
FF
KK
NN
AA
HH
32
GGJJ
YY
DD
LL
MM
KK
SS
FF
TT
RR
AA
QQ
VV
PP
EE
33
AASS
34
VV
SS
JJ
EE
AA
MM
NN
TT
RR
DD
LL
GG
YY
XX
PP
FF
QQ
HH
CC
WW
35
KK
YYNN
FF
EE
MM
SS
RR
AA
GG
LL
QQ
VV
TT
HH
PP
36 37
SS
LL
TT
GG
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38
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39
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40AA
41
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47
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54
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55
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56 57 58 59YY
60 61 62
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64
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65
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66
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67
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68
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69
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70
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71
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72
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73
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74
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75
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MM
LL
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76
SS
KK
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MM
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YYGG
TT
CC 0
1
2
3
4
bits
1
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MM
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QQ
2
SS
PPJJ
II
MM
WW
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3
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4
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14 15
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16
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17
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18
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XX
19 20 21
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22
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24
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26
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27
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28
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29
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30
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31
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32
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35
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36 37
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45
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47
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48
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49
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54
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56
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57 58 59
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60 61 62
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63
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64
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65
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66
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67
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68
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69
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70
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71
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72
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73
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74
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75
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76
RR
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GG
CC 0
1
2
3
4
bits
1
AA
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23
LL
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TT
4
AA
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13
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14 15 16
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17
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18
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19 20 21
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22
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23
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24
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25
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26
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27
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28
SS
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29
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30
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31
EE
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32
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33
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34
GG
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35
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36 37
FF
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RR
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38
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39
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40
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41
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42
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43
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44
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45
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46
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47
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48
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49
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50
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51
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HH
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52
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53
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54
SS
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55
VV
MM
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56 57 58 59 60 61 62
II
AA
LL
JJ
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RR
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GG
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63
DD
TT
KK
VV
RR
HH
FF
EE
AA
QQ
NN
LL
YY
WW
JJ
PP
SS
XX
CC
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64
PPAAEE
65
PP
II
DD
WW
SS
EE
VV
RR
JJ
GG
AA
NN
KK
FF
TT
LL
MM
CC
66
SSLL
XX
KK
II
VV
TT
MM
RR
PP
JJ
NN
WW
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67
AA
GG
EE
VV
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XX
QQ
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LL
RR
SS
68
KKSS
NN
II
MM
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LL
XX
RR
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PP
GG
TT
VV
EE
AA
DD
HH
69
KK
DD
II
RR
WW
TT
NN
MM
LL
SS
JJ
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VV
AA
YY
CC
FF
XX
EE
70
SS
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GG
XX
YY
FF
JJ
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QQ
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LL
MM
AA
VV
NN
II
71
EE
NN
II
VV
KK
QQ
FF
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TT
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LL
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72
AA
GGSS
EE
NN
CC
XX
II
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FF
HH
DD
73
LL
PP
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NN
WW
CC
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II
RR
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AA
74
SS
TT
KK
FF
II
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DD
RR
75
PP
VV
NN
LL
MM
JJ
RR
WW
XXEE
76
RRKKGG
TT
CC 0
1
2
3
4
bits
1
AAMMNN
QQ
2
EEMM
3
WWRR
4
VV
EE
RRWWMM
KK
YY
NN
5
AAII
LL
RR
TT
6
HH
MM
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PP
LL
AA
GG
TT
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7
GG
SSKKRR
TT
AA
WW
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VV
89
VV
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GG
10 11
CCSS
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GG
EE
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12
PPSS
VV
GG
WW
II
MM
RR
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KK
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AA
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13
VVGG
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14
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15 16
MM
NN
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TT
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AA
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XX
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GG
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FF
17
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AA
18XX
PP
19 20 21
CC
WW
TT
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GG
QQ
JJ
NN
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XX
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MM
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FF
II
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AA
HH
22
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MM
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23
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24
LL
25
VVSSYY
26
CC
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27
WWXX
28
LLGG
29SS
30
VVMM
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31
HH
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AA
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VV
NN
32
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33
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34
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35
SS
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36 37
RR
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38
KK
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DD
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39
SS
RR
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KK
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MM
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40 41
MM
VVRR
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42
WW
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NN
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FF
43
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44
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45
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46
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47
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48
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49
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QQ
50
EE
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GG
PP
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XX
MM
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II
WW
51
EEGGDD
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52
RRTT
53
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PP
TT
SS
KK
QQ
YY
54
XX
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55
RR
CC
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56 57 58 59 60 61 62
RREETT
VV
GG
JJ
63
VV
DD
EE
HH
GG
QQ
RR
NN
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FF
MM
YY
II
CC
TT
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WW
XX
LL
AA
PP
64 65
RR
TT
AA
GG
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MM
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XX
CC
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66
NN
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XX
VVPP
WW
QQ
67
LL
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TT
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68
RRLLXXPP
VV
GG
TT
HH
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AA
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69
PPTT
SS
LL
MM
AA
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CC
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70
TTWW
PPAA
MMVV
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71
MM
AA
HH
TT
RR
PP
GG
LL
SS
72
RRLL
PP
MM
HH
FF
DD
73
LLAA
SS
74
MMRR
75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
HHQQ
NN
23
LL
EE
TT
4
RR
JJ
TTNN
WW
KK
MM
YY
5
LL
TT
FF
KK
II
6
NN
VV
MM
LL
PP
TT
RR
QQ
AA
DD
GG
7
RR
KK
AA
JJ
YY
VV
WW
II
89
10 11
QQ
NN
LL
GG
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genera
Oscillatoria
Arthrospira
Anabaena
Nostoc
Cyanothece
Spirulina
clades
Pleurocapsa
Prochlorococcus
Gloeobacter
Pseudanabaena
Acaryochloris
Nodosilinea
function logos
Genome
g
tRNA
genes
T
g
C
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g
Figure 2: Schematic overview of CYANO-MLP phyloclassification workflow. (A) The
cyanobacterial phylogeny from (6) was used to define cyanobacterial clades for which we
estimated tRNA CIFs. Cyanobacterial clades, indicated by background colors, were named
according to (6), except for clade B2+3, which combines clades B2 and B3. Clades with grey
backgrounds were excluded from analysis because of limited genome sample sizes. Next, tRNA
genes are predicted from cyanobacterial genomes and combined by clade for function logo es-
timation (28). (B) Example function logos, for Uracil, with background colors corresponding
to each cyanobacterial clade. (C) For a genomegto be classified, tRNA gene complements
Tgare predicted and scored against the logos for each clade, to produce input score vectors for
CYANO-MLP training and classifications. (D) The architecture of the artificial neural network
CYANO-MLP, and a classification probability vector output from CYANO-MLP represented as
a stacked bar chart.
19
Travis Lawrence
Lawrenceet al. BMC Evolutionary Biology (2019) 19:224
https://doi.org/s12862-019-1552-7
RESEARCH ARTICLE Open Access
tRNAfunctionalsignaturesclassify
plastidsaslate-branchingcyanobacteria
Travis J Lawrence
1,2*
,KatherineCHAmrine
2,3
, Wesley D Swingley
4
and David H Ardell
2,5
Abstract
Background:Eukaryotes acquired the trait of oxygenic photosynthesis through endosymbiosis of the
cyanobacterial progenitor of plastid organelles. Despite recent advances in the phylogenomics of Cyanobacteria, the
phylogenetic root of plastids remains controversial. Although a single origin of plastids by endosymbiosis is broadly
supported, recent phylogenomic studies are contradictory on whether plastids branch early or late within
Cyanobacteria. One underlying cause may be poor fit of evolutionary models to complex phylogenomic data.
Results:Using Posterior Predictive Analysis, we show that recently applied evolutionary models poorly fit three
phylogenomic datasets curated from cyanobacteria and plastid genomes because of heterogeneities in both
substitution processes across sites and of compositions across lineages. To circumvent these sources of bias, we
developed CYANO-MLP, a machine learning algorithm that consistently and accurately phylogenetically classifies
(“phyloclassifies”) cyanobacterial genomes to their clade of origin based on bioinformatically predicted
function-informative features in tRNA gene complements. Classification of cyanobacterial genomes with CYANO-MLP
is accurate and robust to deletion of clades, unbalanced sampling, and compositional heterogeneity in input tRNA
data. CYANO-MLP consistently classifies plastid genomes into a late-branching cyanobacterial sub-clade containing
single-cell, starch-producing, nitrogen-fixing ecotypes, consistent with metabolic and gene transfer data.
Conclusions:Phylogenomic data of cyanobacteria and plastids exhibit both site-process heterogeneities and
compositional heterogeneities across lineages. These aspects of the data require careful modeling to avoid bias in
phylogenomic estimation. Furthermore, we show that amino acid recoding strategies may be insufficient to mitigate
bias from compositional heterogeneities. However, the combination of our novel tRNA-specific strategy with machine
learning in CYANO-MLP appears robust to these sources of bias with high accuracy in phyloclassification of
cyanobacterial genomes. CYANO-MLP consistently classifies plastids as late-branching Cyanobacteria, consistent with
independent evidence from signature-based approaches and some previous phylogenetic studies.
Keywords:Plastids, tRNAs, Cyanobacteria, Primary endosymbiosis, Machine learning
Background
Over one billion years ago [1–3]photosyntheticeukary-
otes originated through endosymbiosis of a cyanobac-
terium with the last common ancestor of Archaeplastida,
aeukaryoticsupergroupencompassinggreenandred
algae, land plants, and glaucophytes [4–6]. The diver-
sity of eukaryotic photoautotrophs radiating from this
event profoundly transformed the terrestrial biosphere
*Correspondence:[email protected]
1
Biosciences Division, Oak Ridge National Laboratory, P.O. Box 2008, 37831
Oak Ridge, TN, USA
2
Quantitative and Systems Biology Program, University of California, Merced,
5200 North Lake Rd., 95343 Merced, CA, USA
Full list of author information is available at the end of the article
through changes to primary biomass production, atmo-
spheric oxygenation, and the colonization of new ecosys-
tems [7,8]. It is widely accepted that plastids originated
in a single primary endosymbiotic event [9]. However,
the phylogenetic root of plastids within Cyanobacteria
remains controversial. Recent phylogenomic studies reach
contradictory conclusions, with plastids branching either
early [10–13] or late [1,14–16]withinCyanobacteriawith
strong statistical support.
Phylogenetic inferences concerning plastid origin are
complicated by large evolutionary distances that have
accumulated over at least one billion years of vertical
descent, by extreme genome reductions in plastids [17]
©TheAuthor(s).2019Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wes SwingleyKatie Amrine
Lawrenceet al. BMC Evolutionary Biology (2019) 19:224
https://doi.org/s12862-019-1552-7
RESEARCH ARTICLE Open Access
tRNAfunctionalsignaturesclassify
plastidsaslate-branchingcyanobacteria
Travis J Lawrence
1,2*
,KatherineCHAmrine
2,3
, Wesley D Swingley
4
and David H Ardell
2,5
Abstract
Background:Eukaryotes acquired the trait of oxygenic photosynthesis through endosymbiosis of the
cyanobacterial progenitor of plastid organelles. Despite recent advances in the phylogenomics of Cyanobacteria, the
phylogenetic root of plastids remains controversial. Although a single origin of plastids by endosymbiosis is broadly
supported, recent phylogenomic studies are contradictory on whether plastids branch early or late within
Cyanobacteria. One underlying cause may be poor fit of evolutionary models to complex phylogenomic data.
Results:Using Posterior Predictive Analysis, we show that recently applied evolutionary models poorly fit three
phylogenomic datasets curated from cyanobacteria and plastid genomes because of heterogeneities in both
substitution processes across sites and of compositions across lineages. To circumvent these sources of bias, we
developed CYANO-MLP, a machine learning algorithm that consistently and accurately phylogenetically classifies
(“phyloclassifies”) cyanobacterial genomes to their clade of origin based on bioinformatically predicted
function-informative features in tRNA gene complements. Classification of cyanobacterial genomes with CYANO-MLP
is accurate and robust to deletion of clades, unbalanced sampling, and compositional heterogeneity in input tRNA
data. CYANO-MLP consistently classifies plastid genomes into a late-branching cyanobacterial sub-clade containing
single-cell, starch-producing, nitrogen-fixing ecotypes, consistent with metabolic and gene transfer data.
Conclusions:Phylogenomic data of cyanobacteria and plastids exhibit both site-process heterogeneities and
compositional heterogeneities across lineages. These aspects of the data require careful modeling to avoid bias in
phylogenomic estimation. Furthermore, we show that amino acid recoding strategies may be insufficient to mitigate
bias from compositional heterogeneities. However, the combination of our novel tRNA-specific strategy with machine
learning in CYANO-MLP appears robust to these sources of bias with high accuracy in phyloclassification of
cyanobacterial genomes. CYANO-MLP consistently classifies plastids as late-branching Cyanobacteria, consistent with
independent evidence from signature-based approaches and some previous phylogenetic studies.
Keywords:Plastids, tRNAs, Cyanobacteria, Primary endosymbiosis, Machine learning
Background
Over one billion years ago [1–3]photosyntheticeukary-
otes originated through endosymbiosis of a cyanobac-
terium with the last common ancestor of Archaeplastida,
aeukaryoticsupergroupencompassinggreenandred
algae, land plants, and glaucophytes [4–6]. The diver-
sity of eukaryotic photoautotrophs radiating from this
event profoundly transformed the terrestrial biosphere
*Correspondence:[email protected]
1
Biosciences Division, Oak Ridge National Laboratory, P.O. Box 2008, 37831
Oak Ridge, TN, USA
2
Quantitative and Systems Biology Program, University of California, Merced,
5200 North Lake Rd., 95343 Merced, CA, USA
Full list of author information is available at the end of the article
through changes to primary biomass production, atmo-
spheric oxygenation, and the colonization of new ecosys-
tems [7,8]. It is widely accepted that plastids originated
in a single primary endosymbiotic event [9]. However,
the phylogenetic root of plastids within Cyanobacteria
remains controversial. Recent phylogenomic studies reach
contradictory conclusions, with plastids branching either
early [10–13] or late [1,14–16]withinCyanobacteriawith
strong statistical support.
Phylogenetic inferences concerning plastid origin are
complicated by large evolutionary distances that have
accumulated over at least one billion years of vertical
descent, by extreme genome reductions in plastids [17]
©TheAuthor(s).2019Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
34
*
66
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221122 15
*
273
Glaucocystophyta
Rhodophyta
Haptophyta
Heterokonta
Cryptophyta
Euglenaceae
Chlorophyta
Charophyta
Bryophyte Monilophytes
Gymnospermae
Nymphaeles
Magnoliids
Monocots
Eudicots
P. chromatophora
G. lithophora
A B1 B2+3 C1 C3 E F G
0
20
40
60
80
100
(C. paradoxa)
Figure 4: CYANO-MLP classification results for genomes of plastids, the chromatophore of
P. chromatophora, and the cyanobacteriumG. lithophora. Row label colors denote Archeap-
lastid clades with Rhodophyta in red, Chloroplastida in green, and Glaucocystophyta in blue,
or non-Archaeplastida in black. Heatmap lower half-cells show average probabilities of clas-
sifications of genomes to clades with text labels denoting percentages of genomes classifying
to cyanobacterial clades. Asterisks (*) denote single genomes. Heatmap upper half-cell colors
(and text labels) show median bootstrap classification frequencies (as percentages). Absence of
labels denotes zero frequencies.
21
Lawrence et al. (2019). BMC Evol. Biol. 19:224
years of divergence between plastids and Cyanobacteria
A B
A
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JJ
56 57 58 59 60 61 62
II
AA
LL
JJ
EE
NN
RR
HH
KK
TT
GG
FF
63
DD
TT
KK
VV
RR
HH
FF
EE
AA
NN
LL
YY
WW
JJ
PP
SS
XX
CC
GG
II
MM
64
PPAAEE
65
PP
II
DD
WW
SS
EE
VV
RR
JJ
GG
AA
NN
KK
FF
TT
LL
MM
CC
66
SSLL
XX
KK
II
VV
TT
MM
RR
PP
JJ
NN
WW
67
AA
GG
EE
VV
TT
XX
WW
YYKK
LL
RR
SS
68
KKSS
NN
II
MM
FFWW
LL
XX
RR
JJ
PP
GG
TT
VV
EE
AA
DD
HH
69
KK
DD
II
RR
WW
TT
NN
MM
LL
SS
JJ
GG
VV
AA
YY
CC
FF
XX
EE
70
SS
EE
DDWW
CCTT
GG
XX
YY
FF
JJ
RR
KK
LL
MM
AA
VV
NN
II
71
EE
NN
II
VV
KK
FF
GG
CC
TT
RR
AA
PP
LL
HH
SS
72
AA
GGSS
EE
NN
CC
XX
II
KK
MM
JJ
RR
TT
PP
FF
HH
DD
73
LL
PP
GGKK
EE
NN
WW
CC
TT
II
RR
MM
VV
JJ
SS
AA
74
SS
TT
KK
FF
II
EE
NN
DD
RR
75
PP
VV
NN
LL
MM
JJ
RR
WW
XXEE
76
RRKKGG
TT
CC 0
1
2
3
4
bits
1
AAMMNN
2
EEMM
3
WWRR
4
VV
EE
RRWWMM
KK
YY
NN
5
AAII
LL
RR
TT
6
HH
MM
DD
PP
LL
AA
GG
TT
RR
JJ
7
GG
SSKKRR
TT
AA
WW
II
VV
89
VV
HH
GG
10 11
CCSS
LL
GG
EE
WW
DD
12
PPSS
VV
GG
WW
II
MM
RR
NN
KK
TT
DD
AA
FF
13
VVGG
EE
14
WWJJ
15 16
MM
NN
KK
TT
VV
PP
DD
RR
AA
XX
WW
CC
SS
HH
LL
GG
JJ
YY
FF
17
EETTDD
RR
LL
MM
WW
II
FF
PP
AA
18XX
PP
19 20 21
CC
WW
TT
VV
GG
JJ
NN
DD
KK
XX
PP
MM
YY
FF
II
SS
LL
AA
HH
22
CCSS
HH
II
YY
MM
RR
DD
23
SS
RREE
24
LL
25
VVSSYY
26
CC
HH
SSJJ
27
WWXX
28
LLGG
29SS
30
VVMM
RR
KK
XX
GG
31
HH
YY
AA
SS
PP
LL
TT
KK
VV
NN
32
LLTT
SS
GG
RR
PP
VV
AA
EE
33
PP
AA
HH
34
VVRR
SS
PP
TT
LL
WW
35
SS
EE
WW
FFAA
RR
LL
VV
GG
CC
TT
HH
MM
PP
36 37
RR
SS
TT
GG
PP
VV
LL
AA
KK
EE
38
KK
YY
DD
NN
HH
EE
39
SS
RR
II
KK
TT
XX
MM
JJ
NN
40 41
MM
VVRR
HH
AA
LL
PP
42
WW
HHVV
TTLL
SS
RR
NN
CC
JJ
YY
FF
43
LLQQ
JJ
44
MMSS
LL
JJ
RR
PP
VV
GG
45
LL
MM
SS
WW
II
CC
46
LL
PP
KK
TT
SS
RR
CC
NN
47
TTVV
GG
HH
LL
SS
KK
YY
NN
CC
48
RR
PP
GG
VV
AA
JJ
WW
FF
49
SS
LL
JJHH
YY
50
EE
LLSS
GG
PP
HH
TT
CC
VV
RR
AA
DD
NN
KK
XX
MM
FF
II
WW
51
EEGGDD
LL
52
RRTT
53
VVLL
RR
PP
TT
SS
KK
YY
54
XX
TT
RRVV
II
YY
55
RR
CC
WWTT
MM
56 57 58 59 60 61 62
RREETT
VV
GG
JJ
63
VV
DD
EE
HH
GG
RR
NN
KK
FF
MM
YY
II
CC
TT
SS
WW
XX
LL
AA
PP
64 65
RR
TT
AA
GG
LL
MM
KK
JJ
XX
CC
FF
66
NN
RR
XX
VVPP
WW
67
LL
PP
EE
CCMM
TT
GG
SS
RR
68
RRLLXXPP
VV
GG
TT
HH
EE
AA
DD
69
PPTT
SS
LL
MM
AA
EE
CC
FF
70
TTWW
PPAA
MMVV
II
71
MM
AA
HH
TT
RR
PP
GG
LL
SS
72
RRLL
PP
MM
HH
FF
DD
73
LLAA
SS
74
MMRR
75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
HHQQ
NN
23
LL
EE
TT
4
RR
JJ
TTNN
WW
KK
MM
YY
5
LL
TT
FF
KK
II
6
NN
VV
MM
LL
PP
TT
RR
AA
DD
GG
7
RR
KK
AA
JJ
YY
VV
WW
II
89
10 11
NN
LL
GG
DD
EE
WW
12
JJ
SSVV
AA
RR
TT
FF
GG
II
DD
KK
WW
MM
NN
13
RR
GG
EE
14 15 16 17
HH
EE
NN
TT
RR
DD
FF
LL
VV
PP
II
WW
AA
18
JJXX
PP
19 20 21
E
J
Q
H
N
LL
T
F
II
C
W
A
G
K
D
Y
S
P
M
V
X
22
VV
MM
JJ
RR
SS
HH
DD
YY
II
23
RR
DDEE
24
RR
LLJJ
25
SSYY
26SS
27
JJXX
28
LL
DD
29 30
SS
RR
KK
JJ
WW
GG
XX
31
AA
VV
SS
YY
TT
GG
PP
LL
FF
KK
NN
HH
32
LLGG
FF
DD
RR
NN
SS
TT
PP
EE
VV
AA
33 34
R
S
T
F
H
DD
KK
CC
PP
WW
35
NN
RR
VV
AA
GG
LL
CC
HH
TT
PP
36 37
SSPP
GG
LL
RR
TT
VV
AA
KK
EE
38
KK
HH
DD
EE
YY
NN
39
SS
RR
TT
XX
MM
KK
NN
JJ
II
40 41
RR
AA
PP
LL
VV
HH
42
AA
EE
RR
NN
TT
JJ
LL
SS
CC
HH
FF
YY
43
RR
JJ
44
VV
RR
SS
PP
LL
GG
45
GG
LL
TT
VV
EE
SS
WW
FF
II
CC
46
LL
JJ
RR
SS
MM
KK
CC
NN
47
JJ
HH
EE
GG
KK
MM
CC
NN
LL
YY
SS
48
JJ
LL
PP
RR
VV
AA
GG
FF
WW
49
JJ
SS
EE
LL
CC
HH
YY
50 51
RR
TT
EE
NN
GG
DD
LL
52 53
RR
JJ
FF
GG
LL
HH
TT
PP
YY
NN
KK
54
PP
RR
JJ
VV
XX
II
YY
55 56 57 58 59 60 61 62
LLTT
JJ
GG
63 64EE
65
RR
AA
GG
DD
LL
TT
CC
66
RR
TT
LL
KK
PP
JJ
WW
NN
67
GG
JJ
LL
WW
RR
SS
XX
68
FF
RR
EE
NN
LL
MM
PP
GG
DD
VV
TT
HH
AA
69
LL
TT
SS
GG
VV
AA
EE
FF
CC
70
RR
SS
AA
JJ
FF
LL
VV
YY
MM
NN
II
71
AA
GG
RR
TT
LL
PP
HH
SS
72
SS
TT
RR
PP
FF
HH
73
RR
JJ
VV
SS
AA
74RR
75
RR
LL
WWEE
76
QQTT
CC
GG 0
1
2
3
4
bits
1
GG
TT
FF
JJ
MM
HH
NN
2
RRFF
HH
CC
3
GG
VV
LL
TT
EE
4
JJ
WW
NN
MM
KK
YY
5
VV
TT
EE
KK
WW
RRII
6
FF
VV
MM
WW
EE
NN
LL
GG
RR
AA
PP
TT
DD
7
GG
AA
KK
VV
WW
JJ
II
89
10
HH
11
RR
LL
GG
EE
DD
WW
12
SS
JJ
WW
VV
RR
KK
AA
GG
TT
II
MM
FF
DD
NN
13
RR
WW
YY
GG
EE
14 15 16
PPKK
JJEE
VV
RR
DD
HH
CC
FF
XX
GG
NN
AA
TT
LL
WW
MM
YY
SS
17
SS
JJ
RR
KK
TT
FF
MM
VV
DD
LL
WW
II
PP
AA
18
VVPP
XX
19 20 21 22
TT
CC
EE
VV
SSDD
HH
YY
RR
MM
II
JJ
23
DD
RR
EE
24
RR
VV
LL
KK
YY
25
SS
FF
NN
YY
26
KKSS
27
VV
EE
WW
QQXX
28
RR
GG
VV
KK
AA
29
VV
LL
30
LL
TT
SS
PP
VV
AA
JJ
MM
CC
RR
WW
GG
XX
31
WW
GG
SS
RR
DD
AA
PP
EE
VV
TT
FF
LL
KK
HH
NN
32
LL
WW
SS
YY
FF
RR
TT
VV
AA
PP
EE
33
VV
FF
34
RR
GG
VV
TT
FF
PP
LL
CC
WW
35
FF
RR
SS
EE
AA
GG
LL
VV
TT
PP
HH
36 37
SS
GG
RR
PP
VV
LL
AA
TT
KK
EE
38
DD
EE
HH
KK
YY
NN
39
SS
RR
II
KK
JJ
TT
XX
NN
MM
40 41
GG
RR
PP
VV
AA
LL
HH
42
VV
KK
EE
AA
MM
NN
RR
LL
TT
FF
HH
YY
JJ
SS
43
GG
RR
CC
JJ
44
TT
KK
JJ
MM
RR
WW
PP
VV
HH
SS
LL
GG
45
LL
SS
GG
TT
WW
FF
CC
II
46
GG
WW
LL
TT
YY
RR
SS
KK
CC
NN
47
TT
RR
GG
VV
WW
EE
HH
LL
YY
NN
KK
SS
CC
48
SS
LL
RR
PP
VV
AA
GG
WW
FF
JJ
49
SS
RR
KK
LL
CC
JJ
YY
HH
50 51
SS
VV
RR
TT
WW
EE
GG
YY
KK
DD
LL
52
VVKK
53
SS
VV
RR
MM
WW
CC
NN
TT
PP
LL
GG
HH
KK
54
RR
NN
VV
XX
YY
II
55 56 57 58 59 60 61 62
LL
KK
RR
TT
HH
YY
NN
GG
FF
63 64
AAEE
65
SS
GG
NN
YY
LL
MM
TT
CC
FF
66
LLGG
FF
SS
VV
RR
MM
YY
TT
KK
PP
NN
JJ
WW
67
GG
VV
LL
SS
RR
XX
68
RR
LL
SS
JJ
MM
FF
GG
PP
VV
EE
TT
DD
HH
AA
69
SS
WW
VV
GG
EE
AA
CC
FF
70
TT
KK
GG
JJ
AA
RR
LL
DD
VV
MM
NN
II
71
FF
JJ
GG
MM
RR
WW
CC
AA
VV
TT
LL
HH
PP
SS
72
GG
TTJJ
MM
RR
PP
HH
FF
DD
73
RR
MM
VV
JJ
SS
AA
74
WW
RR
75
RR
WW
LL
YYEE
76
VV
EE
WW
QQGG
TT
CC 0
1
2
3
4
bits
1
JJ
DDQQ
NN
2HH
3
GG
VV
EE
LL
MM
DD
JJ
TT
4
AA
FF
RR
DD
CC
HH
WW
NN
KK
YY
5
AA
KK
LL
FF
HH
EE
TT
NN
WW
RR
DD
II
6
VV
KK
MM
NN
LL
EE
HH
CC
DD
TT
AA
RR
GG
PP
7
SS
KK
EE
AA
MM
JJ
VV
WW
II
89
10 11
RR
PP
LL
HH
GG
DD
EE
WW
12
PP
SS
KK
JJ
TT
AA
GG
WW
VV
RR
II
MM
NN
FF
DD
13
LL
SS
AAWW
DD
EE
GG
14 15 16
PP
II
EE
MM
KK
TT
XX
NN
DDHH
YY
CC
VV
AA
FF
JJ
LL
GG
RR
SS
WW
17
SS
KK
EE
TT
QQMM
RR
LL
DD
VV
PP
FF
WW
AA
II
18
RRJJ
PP
XX
19 20
DD
RR
21 22
GG
KK
EE
CC
VV
SS
MM
RR
HH
DD
II
YY
23
SSRR
DD
CCEE
24
SS
LL
25
SS
FF
NNYY
26
KKTT
SS
27
EE
JJ
XX
28
LLGG
FF
AA
VV
KK
29
LL
GG
30
LL
EE
PP
WW
SS
RR
AA
TT
JJ
VV
KK
GG
XX
31
PP
RR
EE
TT
JJ
FF
YY
SS
AA
LL
VV
HH
KK
NN
32
SS
YY
CC
LL
RR
FF
VV
AA
TT
PP
GG
EE
33
SS
LL
MM
34
GG
RR
YY
LL
SS
TT
EE
PP
FF
HH
CC
MM
WW
35
SS
RR
EE
AA
LL
GG
HH
VV
TT
PP
36 37
SS
RR
TT
GG
LL
VV
AA
KK
PP
EE
38
KK
YY
NN
EE
HH
DD
39
SS
RR
XX
MM
KK
NN
JJ
TT
II
40 41
RR
AA
PP
VV
LL
HH
42
GG
RR
EE
HH
AA
TT
KK
NN
JJ
LL
FF
SS
YY
MM
43
AA
LL
HH
44
MM
JJ
AA
WW
RR
PP
SS
GG
CC
VV
LL
45
PP
EE
GG
LL
MM
YY
SS
JJ
FF
HH
WW
CC
II
46
EE
GG
VV
LL
TT
SS
RR
NN
JJ
KK
CC
MM
47
VV
MM
HH
WW
GG
NN
CC
LL
YY
SS
KK
48
AA
LL
KK
HH
PP
VV
GG
RR
FF
WW
49
KK
MM
LL
CC
JJ
YY
HH
50
S
LL
CC
EE
JJ
AA
GG
MM
NN
KK
PP
TT
RR
FF
WW
HH
II
VV
XX
DD
51
SS
RR
TT
VV
AA
NN
HH
WW
EE
MM
GG
JJ
KK
DD
LL
52
EE
PP
53
LLAA
EE
GG
RR
YY
CC
TT
II
FF
JJ
NN
KK
MM
HH
PP
54
LL
PP
KK
RR
CC
NN
VV
JJ
XX
YY
II
55 56 57 58 59 60 61 62
SS
TT
LL
KK
RR
PP
NN
FF
MM
HH
GG
JJ
63 64
AAEE
65
SS
RR
KK
EE
NN
DD
JJ
AA
MM
TT
GG
LL
FF
CC
66
SS
VV
TT
JJ
RR
LL
NN
PP
WW
67
PP
WW
JJ
RR
VV
GG
LL
SS
68
SS
KK
RR
II
CC
LL
JJ
EE
FF
PP
VV
GG
AA
TT
HH
DD
XX
69
GGKK
RR
DD
WW
CC
TT
SS
LL
VV
MM
EE
FF
AA
YY
70
WW
CC
AA
LL
RR
VV
FF
KK
NN
II
71
KK
AA
CC
VV
TT
RR
GG
LL
HH
PP
SS
72
EE
GG
RR
JJ
PP
DD
HH
FF
MM
73
PP
JJ
WW
VV
AA
SS
74
VVRR
75
RR
VV
LLMM
EE
76
SS
KKTT
GG
CC 0
1
2
3
4
bits
1NN
23
NNTT
CC
4
RRMM
NN
WW
KK
YY
5
KK
VV
RR
LL
EEII
6
VV
LLRR
DD
GG
PP
AA
TT
7
KK
AA
VV
JJ
II
WW
89
10
SS
YY
11
SS
LL
QQDD
GG
WW
EE
12
JJ
SSVV
TT
RR
DD
GG
FF
AA
WW
II
MM
KK
NN
13
RR
GG
EE
14 15 16
PPKK
NN
MM
HH
AA
VV
RR
JJ
LL
SS
CC
YY
DD
TT
GG
FF
WW
17
RR
NN
VV
LL
TT
WW
DD
AA
FF
II
PP
18
RRJJPP
XX
19 20 21
DD
NN
LL
AA
VV
TT
FF
GG
XX
CC
WW
HH
KK
MM
YY
PP
II
SS
JJ
22
AA
VV
SS
JJ
RR
DD
II
HH
YY
23
SS
RR
EE
24
GG
LLJJ
25
RRSS
26
RR
GGSS
27
JJXX
28
GG
SS
DD
VV
29
RRLL
MM
GG
30
SS
RR
TT
JJ
HH
PP
KK
GG
WW
XX
31
DD
RR
TT
VV
GG
PP
LL
AA
KK
NN
32
LLKK
SS
RR
TT
GG
AA
PP
VV
EE
33
VV
SS
34
TT
LL
GG
PP
SS
CC
WW
35
SSRR
MM
LL
AA
TT
GG
VV
HH
PP
36 37
SS
RR
GG
TT
PP
VV
LL
AA
EE
KK
38
HH
YY
DD
KK
NN
EE
39
SS
RR
II
XX
MM
NN
TT
KK
JJ
40 41
VV
RR
LL
AA
PP
HH
42
RR
KK
SS
AA
NN
TT
LL
JJ
FF
HH
YY
43
PP
RRJJ
44
RR
TT
SS
PP
LL
VV
JJ
GG
45
AA
GG
PP
SS
WW
LL
EE
CC
HH
FF
II
46
TT
GG
JJ
CC
YY
LL
RR
SS
NN
KK
MM
47
RR
GG
HH
CC
MM
SS
LL
NN
KK
YY
48
KK
GG
VV
RR
JJ
FF
WW
49
RR
LL
KKJJ
HH
CC
YY
50
LL
KK
JJ
CCVV
RR
PP
GG
FF
DD
AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
JJ
GG
DD
LL
52 53
RR
KK
LL
NN
PP
TT
GG
SS
HH
YY
54
RR
NN
JJ
VV
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
RRTT
KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
FF
EE
70
RR
VV
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
genera
Oscillatoria
Arthrospira
Anabaena
Nostoc
Cyanothece
Spirulina
clades
Pleurocapsa
Prochlorococcus
Gloeobacter
Pseudanabaena
Acaryochloris
Nodosilinea
function logos
Genome
g
tRNA
genes
T
g
C
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g
Figure 2: Schematic overview of CYANO-MLP phyloclassification workflow. (A) The
cyanobacterial phylogeny from (6) was used to define cyanobacterial clades for which we
estimated tRNA CIFs. Cyanobacterial clades, indicated by background colors, were named
according to (6), except for clade B2+3, which combines clades B2 and B3. Clades with grey
backgrounds were excluded from analysis because of limited genome sample sizes. Next, tRNA
genes are predicted from cyanobacterial genomes and combined by clade for function logo es-
timation (28). (B) Example function logos, for Uracil, with background colors corresponding
to each cyanobacterial clade. (C) For a genomegto be classified, tRNA gene complements
Tgare predicted and scored against the logos for each clade, to produce input score vectors for
CYANO-MLP training and classifications. (D) The architecture of the artificial neural network
CYANO-MLP, and a classification probability vector output from CYANO-MLP represented as
a stacked bar chart.
19
Travis Lawrence
Lawrenceet al. BMC Evolutionary Biology (2019) 19:224
https://doi.org/s12862-019-1552-7
RESEARCH ARTICLE Open Access
tRNAfunctionalsignaturesclassify
plastidsaslate-branchingcyanobacteria
Travis J Lawrence
1,2*
,KatherineCHAmrine
2,3
, Wesley D Swingley
4
and David H Ardell
2,5
Abstract
Background:Eukaryotes acquired the trait of oxygenic photosynthesis through endosymbiosis of the
cyanobacterial progenitor of plastid organelles. Despite recent advances in the phylogenomics of Cyanobacteria, the
phylogenetic root of plastids remains controversial. Although a single origin of plastids by endosymbiosis is broadly
supported, recent phylogenomic studies are contradictory on whether plastids branch early or late within
Cyanobacteria. One underlying cause may be poor fit of evolutionary models to complex phylogenomic data.
Results:Using Posterior Predictive Analysis, we show that recently applied evolutionary models poorly fit three
phylogenomic datasets curated from cyanobacteria and plastid genomes because of heterogeneities in both
substitution processes across sites and of compositions across lineages. To circumvent these sources of bias, we
developed CYANO-MLP, a machine learning algorithm that consistently and accurately phylogenetically classifies
(“phyloclassifies”) cyanobacterial genomes to their clade of origin based on bioinformatically predicted
function-informative features in tRNA gene complements. Classification of cyanobacterial genomes with CYANO-MLP
is accurate and robust to deletion of clades, unbalanced sampling, and compositional heterogeneity in input tRNA
data. CYANO-MLP consistently classifies plastid genomes into a late-branching cyanobacterial sub-clade containing
single-cell, starch-producing, nitrogen-fixing ecotypes, consistent with metabolic and gene transfer data.
Conclusions:Phylogenomic data of cyanobacteria and plastids exhibit both site-process heterogeneities and
compositional heterogeneities across lineages. These aspects of the data require careful modeling to avoid bias in
phylogenomic estimation. Furthermore, we show that amino acid recoding strategies may be insufficient to mitigate
bias from compositional heterogeneities. However, the combination of our novel tRNA-specific strategy with machine
learning in CYANO-MLP appears robust to these sources of bias with high accuracy in phyloclassification of
cyanobacterial genomes. CYANO-MLP consistently classifies plastids as late-branching Cyanobacteria, consistent with
independent evidence from signature-based approaches and some previous phylogenetic studies.
Keywords:Plastids, tRNAs, Cyanobacteria, Primary endosymbiosis, Machine learning
Background
Over one billion years ago [1–3]photosyntheticeukary-
otes originated through endosymbiosis of a cyanobac-
terium with the last common ancestor of Archaeplastida,
aeukaryoticsupergroupencompassinggreenandred
algae, land plants, and glaucophytes [4–6]. The diver-
sity of eukaryotic photoautotrophs radiating from this
event profoundly transformed the terrestrial biosphere
*Correspondence:[email protected]
1
Biosciences Division, Oak Ridge National Laboratory, P.O. Box 2008, 37831
Oak Ridge, TN, USA
2
Quantitative and Systems Biology Program, University of California, Merced,
5200 North Lake Rd., 95343 Merced, CA, USA
Full list of author information is available at the end of the article
through changes to primary biomass production, atmo-
spheric oxygenation, and the colonization of new ecosys-
tems [7,8]. It is widely accepted that plastids originated
in a single primary endosymbiotic event [9]. However,
the phylogenetic root of plastids within Cyanobacteria
remains controversial. Recent phylogenomic studies reach
contradictory conclusions, with plastids branching either
early [10–13] or late [1,14–16]withinCyanobacteriawith
strong statistical support.
Phylogenetic inferences concerning plastid origin are
complicated by large evolutionary distances that have
accumulated over at least one billion years of vertical
descent, by extreme genome reductions in plastids [17]
©TheAuthor(s).2019Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wes SwingleyKatie Amrine
Lawrenceet al. BMC Evolutionary Biology (2019) 19:224
https://doi.org/s12862-019-1552-7
RESEARCH ARTICLE Open Access
tRNAfunctionalsignaturesclassify
plastidsaslate-branchingcyanobacteria
Travis J Lawrence
1,2*
,KatherineCHAmrine
2,3
, Wesley D Swingley
4
and David H Ardell
2,5
Abstract
Background:Eukaryotes acquired the trait of oxygenic photosynthesis through endosymbiosis of the
cyanobacterial progenitor of plastid organelles. Despite recent advances in the phylogenomics of Cyanobacteria, the
phylogenetic root of plastids remains controversial. Although a single origin of plastids by endosymbiosis is broadly
supported, recent phylogenomic studies are contradictory on whether plastids branch early or late within
Cyanobacteria. One underlying cause may be poor fit of evolutionary models to complex phylogenomic data.
Results:Using Posterior Predictive Analysis, we show that recently applied evolutionary models poorly fit three
phylogenomic datasets curated from cyanobacteria and plastid genomes because of heterogeneities in both
substitution processes across sites and of compositions across lineages. To circumvent these sources of bias, we
developed CYANO-MLP, a machine learning algorithm that consistently and accurately phylogenetically classifies
(“phyloclassifies”) cyanobacterial genomes to their clade of origin based on bioinformatically predicted
function-informative features in tRNA gene complements. Classification of cyanobacterial genomes with CYANO-MLP
is accurate and robust to deletion of clades, unbalanced sampling, and compositional heterogeneity in input tRNA
data. CYANO-MLP consistently classifies plastid genomes into a late-branching cyanobacterial sub-clade containing
single-cell, starch-producing, nitrogen-fixing ecotypes, consistent with metabolic and gene transfer data.
Conclusions:Phylogenomic data of cyanobacteria and plastids exhibit both site-process heterogeneities and
compositional heterogeneities across lineages. These aspects of the data require careful modeling to avoid bias in
phylogenomic estimation. Furthermore, we show that amino acid recoding strategies may be insufficient to mitigate
bias from compositional heterogeneities. However, the combination of our novel tRNA-specific strategy with machine
learning in CYANO-MLP appears robust to these sources of bias with high accuracy in phyloclassification of
cyanobacterial genomes. CYANO-MLP consistently classifies plastids as late-branching Cyanobacteria, consistent with
independent evidence from signature-based approaches and some previous phylogenetic studies.
Keywords:Plastids, tRNAs, Cyanobacteria, Primary endosymbiosis, Machine learning
Background
Over one billion years ago [1–3]photosyntheticeukary-
otes originated through endosymbiosis of a cyanobac-
terium with the last common ancestor of Archaeplastida,
aeukaryoticsupergroupencompassinggreenandred
algae, land plants, and glaucophytes [4–6]. The diver-
sity of eukaryotic photoautotrophs radiating from this
event profoundly transformed the terrestrial biosphere
*Correspondence:[email protected]
1
Biosciences Division, Oak Ridge National Laboratory, P.O. Box 2008, 37831
Oak Ridge, TN, USA
2
Quantitative and Systems Biology Program, University of California, Merced,
5200 North Lake Rd., 95343 Merced, CA, USA
Full list of author information is available at the end of the article
through changes to primary biomass production, atmo-
spheric oxygenation, and the colonization of new ecosys-
tems [7,8]. It is widely accepted that plastids originated
in a single primary endosymbiotic event [9]. However,
the phylogenetic root of plastids within Cyanobacteria
remains controversial. Recent phylogenomic studies reach
contradictory conclusions, with plastids branching either
early [10–13] or late [1,14–16]withinCyanobacteriawith
strong statistical support.
Phylogenetic inferences concerning plastid origin are
complicated by large evolutionary distances that have
accumulated over at least one billion years of vertical
descent, by extreme genome reductions in plastids [17]
©TheAuthor(s).2019Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
34
*
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273
Glaucocystophyta
Rhodophyta
Haptophyta
Heterokonta
Cryptophyta
Euglenaceae
Chlorophyta
Charophyta
Bryophyte Monilophytes
Gymnospermae
Nymphaeles
Magnoliids
Monocots
Eudicots
P. chromatophora
G. lithophora
A B1 B2+3 C1 C3 E F G
0
20
40
60
80
100
(C. paradoxa)
Figure 4: CYANO-MLP classification results for genomes of plastids, the chromatophore of
P. chromatophora, and the cyanobacteriumG. lithophora. Row label colors denote Archeap-
lastid clades with Rhodophyta in red, Chloroplastida in green, and Glaucocystophyta in blue,
or non-Archaeplastida in black. Heatmap lower half-cells show average probabilities of clas-
sifications of genomes to clades with text labels denoting percentages of genomes classifying
to cyanobacterial clades. Asterisks (*) denote single genomes. Heatmap upper half-cell colors
(and text labels) show median bootstrap classification frequencies (as percentages). Absence of
labels denotes zero frequencies.
21
Lawrence et al. (2019). BMC Evol. Biol. 19:224
Outline tRNA CIFs Predict Functional
Divergences Between Human and
Trypanosome aaRSs
Rare tRNA CIFs are More
Functionally Informativescores Cyano-MLP
13
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68
II
QQ
RR
SS
NN
MM
KK
JJ
EE
LL
TT
PP
GG
FF
XX
VV
AA
HH
DD
69
KK
QQDDJJHH
RR
LL
MM
SS
NN
TT
AA
WW
YY
VV
GG
EE
FF
CC
XX
70
PPHH
EEVV
CC
SS
QQ
JJ
AA
RR
LL
FF
KK
NN
MM
II
71
GG
EE
KK
NN
QQ
RR
WW
DD
JJ
AA
VV
TT
CC
PP
LL
HH
SS
72
LL
AA
SS
WW
GG
KK
NN
MM
JJ
TT
RR
PP
FF
DD
HH
73
TT
II
RR
KK
MM
NN
QQ
WW
VV
JJ
SS
AA
74
SS
PP
KK
VV
QQ
EE
WW
JJDD
XXRR
75
KK
SS
XX
NN
QQ
VV
WW
RR
JJ
MM
LLEE
76
RR
LLKK
FF
SS
XX
EETT
GG
CC 0
1
2
3
4
bits
1
AA
GG
VV
KKNN
QQ
23
LL
RR
VV
FF
EE
CC
TT
4
AA
GG
VV
LL
CC
DD
HH
TT
JJ
RR
WW
MM
KK
NN
YY
5
LLJJ
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NN
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AA
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DD
RR
WW
II
6
SSII
JJ
CC
MM
KK
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VV
RR
TT
PP
AA
QQ
GG
DD
7
SS
PP
LL
RR
NN
KK
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TT
AA
VV
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WW
II
89
10
SSYY
11
SS
AA
PP
RR
HH
TTLLGG
DD
EE
WW
12
PPEEJJ
SSMM
VV
WW
TT
GG
NN
DD
KK
RR
AA
FF
II
13
SS
KK
JJ
DD
HH
TT
MM
QQ
WW
RR
GG
EE
14 15 16
PP
II
QQ
JJKK
EE
DD
VV
NN
RR
HH
SS
XX
MM
TT
CC
FF
GG
YY
WW
AA
LL
17
SSNN
QQ
CC
EE
YY
JJ
KK
RR
TT
VV
MM
LL
DD
FF
II
PP
WW
AA
18
FFJJXX
PP
19 20 21
R
EETT
KK
WW
LL
NN
QQ
VV
GG
PP
AA
II
SS
YY
JJ
DD
FF
XX
CC
HH
MM
22
TT
PP
GG
EE
CCVV
SS
JJ
MM
RR
HH
II
DD
YY
23
PP
DD
SSRREE
24
LL
MM
JJ
25
TT
PPHH
QQ
SSYY
26
PP
GG
TT
JJ
MMSS
27
AA
PP
KK
EE
JJXX
28
SS
LL
JJ
EE
NN
GG
PP
WW
RR
TT
KK
DD
AA
VV
29
RR
PP
EE
NN
GG
TT
VV
LL
30
PP
LL
II
EE
VV
FF
CC
SS
MMTT
DD
QQ
AA
JJ
KK
RR
WW
GG
XX
31
EE
XX
RR
JJ
IIYY
CC
FF
SS
VV
DD
TT
GG
AA
PP
LL
HH
KK
NN
32
LLGG
II
KK
NN
CC
YY
QQ
FF
SS
DD
RR
TT
AA
PP
VV
EE
33
LL
AA
EE
SS
34
GG
VV
II
RR
AA
JJ
EE
DD
FF
TT
LL
QQ
SS
HH
PP
CC
WW
35
II
KK
EE
MMFF
SS
CC
RR
AA
VV
GG
LL
TT
QQ
PP
HH
36 37
FF
IISS
RR
PP
TT
GG
LL
AA
VV
KK
EE
QQ
38
FFNN
EE
DD
QQ
KK
HH
YY
39
FFSS
RR
JJ
XX
KK
II
NN
MM
TT
40
RRDD
41
KK
GG
MMRR
AA
PP
LL
VV
QQ
HH
42
VV
GG
XXEE
QQ
CC
NN
MM
KK
AA
RR
HH
JJ
TT
SS
LL
FF
YY
43
TT
KK
LL
WW
SS
EE
YY
RR
MM
CC
QQ
JJ
44
TTJJ
YY
WW
HH
KK
CC
VV
MM
AA
SS
RR
PP
LL
GG
45
PP
KK
EE
MM
RR
GG
HH
LL
SS
VV
TT
WW
CC
FF
II
46
PPVV
QQII
YY
GG
LL
MM
TT
SS
RR
JJ
CC
KK
NN
47
II
VV
WW
HH
MM
RR
JJ
TT
GG
CC
LL
NN
KK
SS
YY
48
LL
NN
HH
TT
EE
DD
JJ
KK
SS
RR
PP
VV
AA
GG
FF
WW
49
AA
PPEE
SS
LL
MM
JJ
CC
HH
QQ
YY
50
SS
YY
EE
LL
JJ
CC
GGPP
MM
KK
VV
RR
QQ
TT
WW
AA
II
HH
FF
NN
DD
XX
51
AA
SS
JJ
II
MM
NN
QQ
YY
WW
HH
EE
TT
RR
KK
GG
DD
LL
52
RR
AA
PP
CC
LL
IIKK
53
AACC
MM
XX
II
FFWW
LL
JJVV
RR
YY
GG
SS
TT
PP
KK
HH
NN
QQ
54
SS
TT
GG
RR
LL
EE
NN
FFPP
JJVV
II
YY
XX
55
VV
MM
JJ
56 57 58 59 60 61 62
II
AA
LL
JJ
EE
NN
RR
QQ
HH
KK
TT
GG
FF
63
DD
TT
KK
VV
RR
HH
FF
EE
AA
QQ
NN
LL
YY
WW
JJ
PP
SS
XX
CC
GG
II
MM
64
PPAAEE
65
PP
II
DD
WW
SS
EE
VV
RR
JJ
GG
AA
NN
KK
FF
TT
LL
MM
CC
66
SSLL
XX
KK
II
VV
TT
MM
RR
PP
JJ
NN
WW
QQ
67
AA
GG
EE
VV
TT
XX
QQ
WW
YYKK
LL
RR
SS
68
KKSS
NN
II
MM
FFWW
LL
XX
RR
JJ
PP
GG
TT
VV
EE
AA
DD
HH
69
KK
DD
II
RR
WW
TT
NN
MM
LL
SS
JJ
GG
VV
AA
YY
CC
FF
XX
EE
70
SS
EE
DDWW
CCTT
GG
XX
YY
FF
JJ
RR
QQ
KK
LL
MM
AA
VV
NN
II
71
EE
NN
II
VV
KK
QQ
FF
GG
CC
TT
RR
AA
PP
LL
HH
SS
72
AA
GGSS
EE
NN
CC
XX
II
KK
MM
JJ
RR
TT
PP
FF
HH
DD
73
LL
PP
GGKK
EE
NN
WW
CC
TT
II
RR
MM
VV
JJ
SS
AA
74
SS
TT
KK
FF
II
EE
NN
DD
RR
75
PP
VV
NN
LL
MM
JJ
RR
WW
XXEE
76
RRKKGG
TT
CC 0
1
2
3
4
bits
1
AAMMNN
QQ
2
EEMM
3
WWRR
4
VV
EE
RRWWMM
KK
YY
NN
5
AAII
LL
RR
TT
6
HH
MM
DD
PP
LL
AA
GG
TT
RR
JJ
7
GG
SSKKRR
TT
AA
WW
II
VV
89
VV
HH
GG
10 11
CCSS
LL
GG
EE
WW
DD
12
PPSS
VV
GG
WW
II
MM
RR
NN
KK
TT
DD
AA
FF
13
VVGG
EE
14
WWJJ
15 16
MM
NN
KK
TT
VV
PP
DD
RR
AA
QQ
XX
WW
CC
SS
HH
LL
GG
JJ
YY
FF
17
EETTDD
RR
LL
MM
WW
II
FF
PP
AA
18XX
PP
19 20 21
CC
WW
TT
VV
GG
QQ
JJ
NN
DD
KK
XX
PP
MM
YY
FF
II
SS
LL
AA
HH
22
CCSS
HH
II
YY
MM
RR
DD
23
SS
RREE
24
LL
25
VVSSYY
26
CC
HH
SSJJ
27
WWXX
28
LLGG
29SS
30
VVMM
QQ
RR
KK
XX
GG
31
HH
YY
AA
SS
PP
LL
TT
KK
VV
NN
32
LLTT
SS
GG
RR
PP
VV
AA
EE
33
PP
AA
HH
34
VVRR
SS
PP
TT
LL
WW
35
SS
EE
WW
FFAA
RR
LL
VV
GG
CC
TT
HH
QQ
MM
PP
36 37
RR
SS
TT
GG
PP
VV
LL
AA
KK
QQ
EE
38
KK
QQ
YY
DD
NN
HH
EE
39
SS
RR
II
KK
TT
XX
MM
JJ
NN
40 41
MM
VVRR
HH
AA
LL
PP
QQ
42
WW
HHVV
TTLL
SS
RR
NN
CC
JJ
YY
FF
43
LLQQ
JJ
44
MMSS
LL
JJ
RR
PP
VV
GG
45
LL
MM
SS
WW
II
CC
46
LL
PP
KK
TT
SS
RR
CC
NN
47
TTVV
GG
HH
LL
SS
KK
YY
NN
CC
48
RR
PP
GG
VV
AA
JJ
WW
FF
49
SS
LL
JJHH
YY
QQ
50
EE
LLSS
GG
PP
HH
TT
CC
VV
RR
AA
QQ
DD
NN
KK
XX
MM
FF
II
WW
51
EEGGDD
LL
52
RRTT
53
VVLL
RR
PP
TT
SS
KK
QQ
YY
54
XX
TT
RRVV
II
YY
55
RR
CC
WWTT
MM
56 57 58 59 60 61 62
RREETT
VV
GG
JJ
63
VV
DD
EE
HH
GG
QQ
RR
NN
KK
FF
MM
YY
II
CC
TT
SS
WW
XX
LL
AA
PP
64 65
RR
TT
AA
GG
LL
MM
KK
JJ
XX
CC
FF
66
NN
RR
XX
VVPP
WW
QQ
67
LL
PP
EE
CCMM
TT
GG
SS
RR
68
RRLLXXPP
VV
GG
TT
HH
EE
AA
DD
69
PPTT
SS
LL
MM
AA
EE
CC
FF
70
TTWW
PPAA
MMVV
II
71
MM
AA
HH
TT
RR
PP
GG
LL
SS
72
RRLL
PP
MM
HH
FF
DD
73
LLAA
SS
74
MMRR
75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
HHQQ
NN
23
LL
EE
TT
4
RR
JJ
TTNN
WW
KK
MM
YY
5
LL
TT
FF
KK
II
6
NN
VV
MM
LL
PP
TT
RR
QQ
AA
DD
GG
7
RR
KK
AA
JJ
YY
VV
WW
II
89
10 11
QQ
NN
LL
GG
DD
EE
WW
12
JJ
SSVV
AA
RR
TT
FF
GG
II
DD
KK
WW
MM
NN
13
RR
QQ
GG
EE
14 15 16 17
HH
EE
NN
TT
QQ
RR
DD
FF
LL
VV
PP
II
WW
AA
18
JJXX
PP
19 20 21
E
J
Q
H
N
LL
T
F
II
C
W
A
G
K
D
Y
S
P
M
V
X
22
VV
MM
JJ
RR
SS
HH
DD
YY
II
23
RR
DDEE
24
RR
LLJJ
25
SSYY
26SS
27
QQ
JJXX
28
LL
QQ
DD
29 30
SS
QQ
RR
KK
JJ
WW
GG
XX
31
AA
VV
SS
YY
TT
GG
PP
LL
FF
KK
NN
HH
32
LLGG
FF
QQ
DD
RR
NN
SS
TT
PP
EE
VV
AA
33 34
R
S
T
F
H
DD
KK
CC
PP
WW
35
NN
RR
VV
QQ
AA
GG
LL
CC
HH
TT
PP
36 37
SSPP
GG
LL
RR
TT
VV
AA
KK
QQ
EE
38
KK
HH
DD
EE
YY
NN
QQ
39
SS
RR
TT
XX
MM
KK
NN
JJ
II
40 41
RR
AA
PP
LL
VV
QQ
HH
42
AA
EE
RR
QQ
NN
TT
JJ
LL
SS
CC
HH
FF
YY
43
RR
QQ
JJ
44
VV
RR
SS
PP
LL
GG
45
GG
LL
TT
VV
EE
SS
WW
FF
II
CC
46
LL
JJ
QQ
RR
SS
MM
KK
CC
NN
47
JJ
HH
EE
GG
KK
MM
CC
NN
LL
YY
SS
48
JJ
LL
PP
RR
VV
AA
GG
FF
WW
49
JJ
SS
EE
LL
CC
QQ
HH
YY
50 51
RR
TT
EE
NN
GG
QQ
DD
LL
52 53
RR
JJ
FF
GG
LL
HH
TT
PP
YY
NN
KK
QQ
54
PP
RR
JJ
VV
XX
II
YY
55 56 57 58 59 60 61 62
LLTT
JJ
GG
63 64EE
65
RR
AA
GG
DD
LL
TT
CC
66
RR
TT
LL
KK
PP
JJ
WW
QQ
NN
67
GG
JJ
LL
WW
RR
SS
XX
68
FF
RR
EE
NN
LL
MM
PP
GG
DD
VV
TT
HH
AA
69
LL
TT
SS
GG
VV
AA
EE
FF
CC
70
RR
SS
AA
JJ
FF
QQ
LL
VV
YY
MM
NN
II
71
AA
GG
RR
TT
LL
PP
HH
SS
72
SS
TT
RR
PP
FF
HH
73
RR
JJ
VV
SS
AA
74RR
75
RR
LL
WWEE
76
QQTT
CC
GG 0
1
2
3
4
bits
1
GG
TT
FF
JJ
MM
HH
QQ
NN
2
RRFF
HH
CC
3
GG
VV
QQ
LL
TT
EE
4
JJ
WW
NN
MM
KK
YY
5
VV
TT
EE
KK
WW
RRII
6
FF
VV
MM
WW
EE
NN
LL
QQ
GG
RR
AA
PP
TT
DD
7
GG
AA
KK
VV
WW
JJ
II
89
10
HH
11
RR
LL
QQ
GG
EE
DD
WW
12
SS
JJ
WW
VV
RR
KK
AA
GG
TT
II
MM
FF
DD
NN
13
QQ
RR
WW
YY
GG
EE
14 15 16
PPKK
JJEE
QQ
VV
RR
DD
HH
CC
FF
XX
GG
NN
AA
TT
LL
WW
MM
YY
SS
17
SS
JJ
RR
KK
QQ
TT
FF
MM
VV
DD
LL
WW
II
PP
AA
18
VVPP
XX
19 20 21 22
TT
CC
EE
VV
SSDD
HH
YY
RR
MM
II
JJ
23
DD
RR
EE
24
RR
VV
LL
KK
YY
25
SS
FF
NN
YY
26
KKSS
27
VV
EE
WW
QQXX
28
RR
GG
VV
KK
QQ
AA
29
VV
LL
QQ
30
LL
TT
SS
PP
VV
AA
JJ
MM
QQ
CC
RR
WW
GG
XX
31
WW
GG
SS
RR
DD
AA
PP
EE
VV
TT
FF
LL
KK
HH
NN
32
LL
WW
SS
YY
FF
RR
TT
QQ
VV
AA
PP
EE
33
VV
FF
34
RR
GG
VV
TT
QQ
FF
PP
LL
CC
WW
35
FF
RR
SS
EE
AA
QQ
GG
LL
VV
TT
PP
HH
36 37
SS
GG
RR
PP
VV
LL
AA
TT
KK
QQ
EE
38
DD
EE
HH
KK
YY
NN
QQ
39
SS
RR
II
KK
JJ
TT
XX
NN
MM
40 41
GG
RR
PP
VV
AA
LL
QQ
HH
42
VV
KK
EE
AA
MM
QQ
NN
RR
LL
TT
FF
HH
YY
JJ
SS
43
GG
RR
CC
JJ
44
TT
KK
JJ
MM
RR
WW
PP
VV
HH
SS
LL
GG
45
LL
SS
GG
TT
WW
FF
CC
II
46
GG
WW
LL
TT
YY
RR
SS
KK
CC
NN
47
TT
RR
GG
VV
WW
EE
HH
LL
YY
NN
KK
SS
CC
48
SS
LL
RR
PP
VV
AA
GG
WW
FF
JJ
49
SS
RR
KK
LL
CC
JJ
QQ
YY
HH
50 51
SS
VV
RR
TT
QQ
WW
EE
GG
YY
KK
DD
LL
52
VVKK
53
SS
VV
RR
MM
WW
CC
NN
TT
PP
LL
QQ
GG
HH
KK
54
RR
NN
VV
XX
YY
II
55 56 57 58 59 60 61 62
LL
KK
RR
TT
HH
YY
NN
GG
FF
63 64
AAEE
65
SS
GG
NN
YY
LL
MM
TT
CC
FF
66
LLGG
FF
SS
VV
RR
MM
YY
TT
KK
PP
NN
JJ
WW
QQ
67
GG
VV
LL
QQ
SS
RR
XX
68
RR
LL
SS
JJ
MM
FF
GG
PP
VV
EE
TT
DD
HH
AA
69
SS
WW
VV
GG
EE
AA
CC
FF
70
TT
KK
GG
JJ
AA
QQ
RR
LL
DD
VV
MM
NN
II
71
FF
JJ
GG
MM
RR
WW
CC
AA
VV
TT
LL
HH
PP
SS
72
GG
TTJJ
MM
RR
PP
HH
FF
DD
73
RR
MM
VV
JJ
SS
AA
74
WW
RR
75
RR
WW
LL
YYEE
76
VV
EE
WW
QQGG
TT
CC 0
1
2
3
4
bits
1
JJ
DDQQ
NN
2HH
3
GG
VV
EE
LL
MM
DD
JJ
TT
4
AA
FF
RR
DD
CC
HH
WW
NN
KK
YY
5
AA
KK
LL
FF
HH
EE
TT
NN
WW
RR
DD
II
6
VV
KK
MM
NN
LL
QQ
EE
HH
CC
DD
TT
AA
RR
GG
PP
7
SS
KK
EE
AA
MM
JJ
VV
WW
II
89
10 11
RR
PP
LL
HH
GG
DD
EE
WW
12
PP
SS
KK
JJ
TT
AA
GG
WW
VV
RR
II
MM
NN
FF
DD
13
LL
SS
AAWW
DD
EE
GG
14 15 16
PP
II
EE
MM
KK
TT
XX
NN
QQ
DDHH
YY
CC
VV
AA
FF
JJ
LL
GG
RR
SS
WW
17
SS
KK
EE
TT
QQMM
RR
LL
DD
VV
PP
FF
WW
AA
II
18
RRJJ
PP
XX
19 20
DD
RR
21 22
GG
KK
EE
CC
VV
SS
MM
RR
HH
DD
II
YY
23
SSRR
DD
CCEE
24
SS
LL
25
SS
FF
NNYY
26
KKTT
SS
27
QQ
EE
JJ
XX
28
LLGG
FF
AA
VV
KK
29
LL
GG
30
LL
EE
PP
QQ
WW
SS
RR
AA
TT
JJ
VV
KK
GG
XX
31
PP
RR
EE
TT
JJ
FF
YY
SS
AA
LL
VV
HH
KK
NN
32
SS
YY
CC
LL
RR
FF
QQ
VV
AA
TT
PP
GG
EE
33
SS
LL
MM
34
GG
RR
QQ
YY
LL
SS
TT
EE
PP
FF
HH
CC
MM
WW
35
SS
RR
EE
AA
LL
GG
QQ
HH
VV
TT
PP
36 37
SS
RR
TT
GG
LL
VV
AA
KK
PP
QQ
EE
38
KK
YY
NN
EE
QQ
HH
DD
39
SS
RR
XX
MM
KK
NN
JJ
TT
II
40 41
RR
AA
PP
VV
LL
QQ
HH
42
GG
RR
EE
HH
AA
TT
KK
NN
JJ
LL
FF
SS
YY
MM
43
AA
LL
HH
44
QQ
MM
JJ
AA
WW
RR
PP
SS
GG
CC
VV
LL
45
PP
EE
GG
LL
MM
YY
SS
JJ
FF
HH
WW
CC
II
46
EE
GG
VV
LL
TT
SS
RR
NN
JJ
KK
CC
MM
47
VV
MM
HH
WW
QQ
GG
NN
CC
LL
YY
SS
KK
48
AA
LL
KK
HH
PP
VV
GG
RR
FF
WW
49
KK
MM
LL
CC
JJ
YY
HH
QQ
50
S
LL
CC
EE
JJ
AA
GG
QQ
MM
NN
KK
PP
TT
RR
FF
WW
HH
II
VV
XX
DD
51
SS
RR
TT
VV
AA
NN
HH
WW
EE
QQ
MM
GG
JJ
KK
DD
LL
52
EE
PP
53
LLAA
EE
GG
QQ
RR
YY
CC
TT
II
FF
JJ
NN
KK
MM
HH
PP
54
LL
PP
KK
RR
CC
NN
VV
JJ
XX
YY
II
55 56 57 58 59 60 61 62
SS
TT
LL
KK
RR
PP
NN
QQ
FF
MM
HH
GG
JJ
63 64
AAEE
65
SS
RR
KK
EE
NN
DD
JJ
AA
MM
TT
GG
LL
FF
CC
66
SS
VV
TT
JJ
RR
LL
NN
PP
WW
QQ
67
PP
WW
JJ
RR
VV
GG
LL
SS
QQ
68
SS
KK
RR
II
CC
LL
JJ
EE
FF
PP
VV
GG
AA
TT
HH
DD
XX
69
GGKK
RR
DD
WW
CC
TT
SS
LL
VV
MM
EE
FF
AA
YY
70
WW
CC
AA
LL
RR
QQ
VV
FF
KK
NN
II
71
KK
QQ
AA
CC
VV
TT
RR
GG
LL
HH
PP
SS
72
EE
GG
RR
JJ
PP
DD
HH
FF
MM
73
PP
JJ
WW
VV
AA
SS
74
VVRR
75
RR
VV
LLMM
EE
76
SS
KKTT
GG
CC 0
1
2
3
4
bits
1NN
QQ
23
NNTT
CC
4
RRMM
NN
WW
KK
YY
5
KK
VV
RR
LL
EEII
6
VV
LLRR
DD
GG
PP
AA
TT
7
KK
AA
VV
JJ
II
WW
89
10
SS
YY
11
SS
LL
QQDD
GG
WW
EE
12
JJ
SSVV
TT
RR
DD
GG
FF
AA
WW
II
MM
KK
NN
13
RR
QQ
GG
EE
14 15 16
PPKK
NN
MM
HH
QQ
AA
VV
RR
JJ
LL
SS
CC
YY
DD
TT
GG
FF
WW
17
RR
QQ
NN
VV
LL
TT
WW
DD
AA
FF
II
PP
18
RRJJPP
XX
19 20 21
DD
QQ
NN
LL
AA
VV
TT
FF
GG
XX
CC
WW
HH
KK
MM
YY
PP
II
SS
JJ
22
AA
VV
SS
JJ
RR
DD
II
HH
YY
23
SS
RR
EE
24
GG
LLJJ
25
RRSS
26
RR
GGSS
27
JJXX
28
GG
SS
DD
VV
29
RRLL
MM
GG
30
SS
RR
TT
QQ
JJ
HH
PP
KK
GG
WW
XX
31
DD
RR
TT
VV
GG
PP
LL
AA
KK
NN
32
LLKK
QQ
SS
RR
TT
GG
AA
PP
VV
EE
33
VV
SS
34
TT
LL
GG
PP
SS
CC
WW
35
SSRR
MM
LL
AA
TT
GG
VV
HH
QQ
PP
36 37
SS
RR
GG
TT
PP
VV
LL
AA
EE
QQ
KK
38
HH
YY
QQ
DD
KK
NN
EE
39
SS
RR
II
XX
MM
NN
TT
KK
JJ
40 41
VV
RR
LL
AA
PP
HH
QQ
42
RR
KK
SS
AA
NN
TT
LL
JJ
FF
HH
YY
43
PP
RRJJ
44
RR
TT
SS
QQ
PP
LL
VV
JJ
GG
45
AA
GG
PP
SS
WW
LL
EE
CC
HH
FF
II
46
TT
GG
JJ
CC
YY
LL
RR
SS
NN
KK
MM
47
RR
GG
HH
CC
MM
SS
LL
NN
KK
YY
48
KK
GG
VV
RR
JJ
FF
WW
49
RR
LL
KKJJ
QQ
HH
CC
YY
50
LL
KK
JJ
CCVV
RR
QQ
PP
GG
FF
DD
AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
QQ
JJ
GG
DD
LL
52 53
RR
KK
LL
QQ
NN
PP
TT
GG
SS
HH
YY
54
RR
NN
JJ
VV
QQ
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
RRTT
KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
QQ
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
QQ
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
QQ
FF
EE
70
RR
VV
QQ
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
function logos
Genome
g
tRNA
genes
T
g
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g tRNA CIFs are Nonetheless Highly
Evolutionarily Conserved tRNA Class Informative Features
(CIFs) are Based on Physics, not
Evolutionary Conservation
Divergences Between Human and
Trypanosome aaRSs
Rare tRNA CIFs are More
Functionally Informativescores Cyano-MLP
13
0.0
1.0
classification
probabilities0
1
2
3
4
bits
1
AA
SSMM
XX
JJ
FFNN
2
GG
JJ
CC
WW
YY
HH
3
GG
NN
VV
LL
CC
TT
EE
4
TT
JJ
EE
VV
AA
CC
RR
WW
HH
NN
MM
KK
YY
5
LL
GGKK
TT
JJ
FF
NN
AA
RR
YY
WW
II
6
VV
EE
MM
YY
JJ
KK
NN
HH
LL
RR
GG
AA
PP
DD
TT
7
SS
RR
GGNN
YY
KK
AA
VV
WW
JJ
II
89
GG
JJ
FF
10
SS
RRYY
11
RR
AA
NN
LL
TT
PP
YY
HHDD
EE
GG
WW
12
PP
JJSSWW
KK
VV
GG
MM
TT
NN
RR
AA
II
FF
DD
13
KK
YY
JJ
LL
CC
TT
AA
RR
MM
WW
GG
EE
14
SS
AA
YY
15
KK
QQJJ
16
PP
II
EE
KK
XX
HH
RR
SSNN
JJ
DD
WW
YY
VV
AA
TT
GG
FF
CC
MM
LL
17
SS
YY
HH
EE
NN
KK
TT
VV
RR
DD
FF
LL
WW
PP
AA
II
18
SS
TT
EEXX
PP
19 20 21
EE
JJ
YYLLDD
WW
KK
AA
TT
SS
MM
HH
XX
NN
FF
GG
PP
II
CC
VV
22
GG
PP
FFWW
NN
XX
CC
KK
EE
JJ
VV
SS
MM
HH
YY
DD
RR
II
23
PP
DD
NN
SS
RREE
24
YY
MM
JJ
LL
KK
25
KK
NN
FF
SS
QQYY
26
TTYY
KK
SS
27
MM
YYXX
28
SS
JJ
FF
EE
MM
VV
LL
AA
YY
TT
WW
KK
GG
RR
29
RR
LL
MM
NN
GG
WW
30
PP
VV
SS
LL
YY
JJ
FF
EE
WW
AA
MM
NNKK
TT
RR
CC
GG
XX
31
RRYY
WW
JJ
CC
EE
PP
TT
LL
DD
SS
GG
VV
FF
KK
NN
AA
HH
32
GGJJ
YY
DD
LL
MM
KK
SS
FF
TT
RR
AA
VV
PP
EE
33
AASS
34
VV
SS
JJ
EE
AA
MM
NN
TT
RR
DD
LL
GG
YY
XX
PP
FF
HH
CC
WW
35
KK
YYNN
FF
EE
MM
SS
RR
AA
GG
LL
VV
TT
HH
PP
36 37
SS
LL
TT
GG
PP
VV
RR
AA
KK
EE
38
NN
EE
HH
DD
YY
KK
39
SS
RR
KK
TT
XX
MM
II
JJ
NN
40AA
41
SSGG
RR
AA
PP
LL
VV
HH
42
NN
GG
EE
RR
AA
KK
HH
TT
YY
FF
LL
SS
JJ
43
TT
GG
LL
EE
DD
NN
AA
SS
MM
RR
YY
CC
JJ
44
FF
AA
NN
HH
JJ
KK
XX
MM
YY
RR
PP
VV
SS
LL
GG
45
KK
GG
PP
DD
MM
HH
RR
SS
EE
LL
YY
TT
FF
CC
WW
II
46
JJ
FF
MM
GG
YY
RR
WW
TT
CC
LL
SS
KK
NN
47
AAQQ
FF
DDTT
WW
RR
JJ
EE
GG
MM
HH
CC
LL
YY
KK
NN
SS
48
TT
KK
HH
SS
NN
AA
JJ
LL
CC
MM
PP
VV
GG
RR
FF
WW
49
RR
KK
FF
EE
SS
JJ
CC
HH
LL
YY
50
YY
JJ
SS
LL
EE
CC
GGQQMM
WW
AA
KK
RR
FF
TT
NN
VV
PP
XX
II
DD
HH
51
SS
PP
FF
CC
HH
JJ
VV
YY
TT
AA
NN
RR
MM
WW
EE
GG
KK
DD
LL
52
GG
II
KK
53
VV
JJ
AA
CC
MM
LL
KK
GG
RR
WW
YY
TT
SS
FF
PP
HH
NN
54
TT
MM
LL
PP
NN
JJ
VV
XX
YY
II
55
GG
NN
YY
56 57 58 59YY
60 61 62
AA
RRDD
EE
CC
XX
KK
LL
MM
YY
FF
NN
TT
GG
HH
JJ
63
DD
FF
TT
YY
AA
HH
NN
RR
MM
EE
JJ
LL
KK
VV
GG
II
XX
SS
CC
PP
WW
64
VVYYEE
65
RR
VV
SS
NN
AA
GG
YY
XX
DD
WW
FF
TT
LL
CC
66
AA
SS
YY
FF
MM
XX
TT
LL
JJ
VV
RR
KK
PP
WW
NN
67
GG
MM
KK
YY
WW
LL
SS
RR
68
SS
NNMM
FF
KK
LL
JJ
GG
PP
TT
EE
XX
VV
AA
HH
DD
69
RRSS
JJ
TT
MMLL
KK
DD
NN
WW
YY
VV
GG
XX
AA
EE
FF
CC
70
KK
SS
GG
WW
NN
LL
RR
JJ
DD
AA
MM
FF
VV
II
71
YY
FF
NN
KK
JJ
WW
AA
CC
TT
GG
RR
LL
VV
HH
PP
SS
72
SS
YY
TT
GG
AA
NN
XXKK
JJ
RR
PP
MM
HH
FF
DD
73
RRTT
NN
JJ
WW
YY
VV
AA
SS
74
YY
DD
NN
XX
WW
RR
75
TT
GGWW
NN
VV
RR
MM
LL
YY
XX
EE
76
SS
KK
JJ
MM
EE
YYGG
TT
CC 0
1
2
3
4
bits
1
RR
PP
SS
LL
KK
EE
FF
MM
JJ
XXNN
2
SS
PPJJ
II
MM
WW
CC
HH
3
SS
VV
EE
RR
FF
JJ
CC
II
MM
LL
TT
4
LL
SS
FF
WW
AA
II
VV
CC
RR
HH
JJNN
MM
KK
YY
5
PP
SS
DD
HH
GG
KK
RR
LL
NN
EE
AA
FFWW
II
6
SS
WW
CC
KK
NN
MM
HH
EE
JJ
VV
LL
RR
GG
PP
TT
AA
DD
7
LL
RR
TT
YY
SS
NN
MM
KK
VV
AA
WW
JJ
II
89
10
WWSSYY
11
VV
SS
LL
HH
PP
RR
QQEE
GG
DD
WW
12
EE
CC
PP
SS
JJ
WW
TT
MM
RR
KK
GG
VV
FF
AA
II
NN
DD
13
PP
AA
SSNN
DD
CC
KK
MM
JJ
YY
RR
WW
GG
EE
14 15
AA
SS
NN
YY
KK
MM
XX
JJ
16
II
PP
XX
EE
KK
JJ
WW
LL
MM
HH
RR
NN
YY
DD
VV
SS
TT
AA
GG
CC
FF
17
SS
TTNN
EE
KK
YY
HH
JJ
RR
MM
WW
VV
DD
FF
II
LL
PP
AA
18
TT
II
FF
JJ
YYMMPP
XX
19 20 21
EE
LL
JJ
NN
WW
MM
KK
AA
DD
SS
GG
TT
PP
CC
YY
VV
XX
FF
II
HH
22
LL
GG
KK
WW
AA
XX
CC
EEVV
SS
JJ
MM
RR
HH
YY
II
DD
23
LLPP
SS
DD
YY
RR
EE
24
SSRR
JJ
YY
KK
LL
25
QQFF
SSYY
26
VV
YYKK
TT
SS
27
JJMM
EEXX
28
PP
TTEE
YYCC
DD
AA
RR
LL
VV
SS
JJ
WW
MM
GG
KK
29
RR
SS
PP
VV
WW
KK
LL
GG
30
LL
NN
JJ
AA
FF
DD
MM
EE
KK
CC
SS
WW
RR
TT
GG
XX
31
MM
II
FFQQ
WW
RR
SS
TT
DD
CC
JJ
EE
PP
AA
GG
VV
LL
KK
HH
NN
32
WW
JJ
YY
HH
IIGG
DD
SS
LL
KK
TT
RR
FF
VV
PP
AA
EE
33
LL
GG
EE
SS
34
AA
MM
KK
SS
VVGG
XX
RR
DD
EE
LL
YY
FF
PP
HH
WW
CC
35
KK
II
NN
CC
MM
XX
WW
DD
EE
FF
SS
RR
VV
AA
GG
LL
TT
HH
PP
36 37
IISS
RR
LL
TT
PP
GG
VV
KK
AA
EE
38
YY
NN
DD
HH
KK
EE
39
SS
RR
MM
XX
TT
JJ
KK
NN
II
40DD
41
SS
EEGG
AA
RR
PP
VV
LL
HH
42
PP
XX
II
WW
EE
MM
AA
RR
HH
KK
NN
LL
TT
FF
JJ
YY
SS
43
II
KK
FF
CC
RR
HH
SS
MM
JJ
44
AA
CC
HH
WW
YY
MM
KK
JJ
XX
SS
PP
RR
VV
LL
GG
45
KK
NN
EE
RR
PP
JJ
VV
GG
LL
SS
HH
TT
MM
FF
WW
CC
II
46
PP
II
VV
EE
DDLL
TT
WW
GG
MM
CC
SS
JJ
RR
YY
KK
NN
47
TTMM
RR
HH
JJ
WW
GG
CC
EE
LL
NN
KK
YY
SS
48
IITT
YY
XX
DD
NN
CC
MM
LL
KK
RR
PP
JJ
SS
GG
VV
AA
WW
FF
49
PP
II
RR
KK
SS
MMJJ
LL
HH
CC
YY
50
SS
EE
YY
CC
LL
JJ
PP
GG
MM
KK
RR
WW
TT
HH
XX
DD
FF
NN
AA
II
VV
51
PP
CCHH
SS
VV
FF
AA
TT
MM
EE
YY
RR
JJ
NN
WW
GG
KK
DD
LL
52
LL
AASS
PP
53
EE
YY
AA
LL
XXCC
DD
SS
RR
JJ
WW
GG
VV
FF
KK
PP
NN
TT
MM
HH
54
SS
TT
FF
WW
LL
JJ
PP
NN
MM
VV
YY
XX
II
55
SSJJMM
56
LL
VV
WW
57 58 59
SS
RR
60 61 62
RR
DD
PP
SS
VV
AA
NN
YY
MM
EE
CC
KK
HH
LL
JJ
FF
GG
TT
63
DD
TT
RR
FF
KK
HH
AA
CC
YY
WW
SS
NN
PP
LL
GG
JJ
EE
VV
MM
XX
II
64
LL
YY
AAEE
65
PP
JJ
HHDD
XX
RR
GG
YY
NN
KK
EE
MM
SS
VV
AA
CC
LL
TT
FF
66
EE
FF
II
SS
XX
TT
MM
VV
YY
KK
LL
RR
PP
NN
JJ
WW
67
AA
PP
YY
MM
WW
XX
KK
LL
SS
RR
68
II
RR
SS
NN
MM
KK
JJ
EE
LL
TT
PP
GG
FF
XX
VV
AA
HH
DD
69
KK
QQDDJJHH
RR
LL
MM
SS
NN
TT
AA
WW
YY
VV
GG
EE
FF
CC
XX
70
PPHH
EEVV
CC
SS
JJ
AA
RR
LL
FF
KK
NN
MM
II
71
GG
EE
KK
NN
RR
WW
DD
JJ
AA
VV
TT
CC
PP
LL
HH
SS
72
LL
AA
SS
WW
GG
KK
NN
MM
JJ
TT
RR
PP
FF
DD
HH
73
TT
II
RR
KK
MM
NN
WW
VV
JJ
SS
AA
74
SS
PP
KK
VV
EE
WW
JJDD
XXRR
75
KK
SS
XX
NN
VV
WW
RR
JJ
MM
LLEE
76
RR
LLKK
FF
SS
XX
EETT
GG
CC 0
1
2
3
4
bits
1
AA
GG
VV
KKNN
23
LL
RR
VV
FF
EE
CC
TT
4
AA
GG
VV
LL
CC
DD
HH
TT
JJ
RR
WW
MM
KK
NN
YY
5
LLJJ
EE
NN
YY
KK
AA
TT
MM
DD
RR
WW
II
6
SSII
JJ
CC
MM
KK
EE
WW
HHLL
VV
RR
TT
PP
AA
GG
DD
7
SS
PP
LL
RR
NN
KK
QQMM
TT
AA
VV
JJ
WW
II
89
10
SSYY
11
SS
AA
PP
RR
HH
TTLLGG
DD
EE
WW
12
PPEEJJ
SSMM
VV
WW
TT
GG
NN
DD
KK
RR
AA
FF
II
13
SS
KK
JJ
DD
HH
TT
MM
WW
RR
GG
EE
14 15 16
PP
II
JJKK
EE
DD
VV
NN
RR
HH
SS
XX
MM
TT
CC
FF
GG
YY
WW
AA
LL
17
SSNN
CC
EE
YY
JJ
KK
RR
TT
VV
MM
LL
DD
FF
II
PP
WW
AA
18
FFJJXX
PP
19 20 21
R
EETT
KK
WW
LL
NN
VV
GG
PP
AA
II
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YY
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22
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YY
23
PP
DD
SSRREE
24
LL
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25
TT
PPHH
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26
PP
GG
TT
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MMSS
27
AA
PP
KK
EE
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28
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LL
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NN
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AA
VV
29
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PP
EE
NN
GG
TT
VV
LL
30
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LL
II
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VV
FF
CC
SS
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DD
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XX
31
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32
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PP
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EE
33
LL
AA
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34
GG
VV
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WW
35
II
KK
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SS
CC
RR
AA
VV
GG
LL
TT
PP
HH
36 37
FF
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LL
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38
FFNN
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39
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40
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41
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42
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44
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45
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47
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48
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49
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50
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52
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53
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NN
54
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YY
XX
55
VV
MM
JJ
56 57 58 59 60 61 62
II
AA
LL
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EE
NN
RR
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TT
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FF
63
DD
TT
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VV
RR
HH
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NN
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CC
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64
PPAAEE
65
PP
II
DD
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AA
NN
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FF
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66
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67
AA
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68
KKSS
NN
II
MM
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PP
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69
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70
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71
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72
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XX
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HH
DD
73
LL
PP
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AA
74
SS
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RR
75
PP
VV
NN
LL
MM
JJ
RR
WW
XXEE
76
RRKKGG
TT
CC 0
1
2
3
4
bits
1
AAMMNN
2
EEMM
3
WWRR
4
VV
EE
RRWWMM
KK
YY
NN
5
AAII
LL
RR
TT
6
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MM
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7
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89
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10 11
CCSS
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12
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13
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14
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15 16
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18XX
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19 20 21
CC
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AA
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22
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23
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24
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25
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26
CC
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27
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28
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29SS
30
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RR
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31
HH
YY
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SS
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32
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33
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34
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35
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CC
TT
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MM
PP
36 37
RR
SS
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38
KK
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NN
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39
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RR
II
KK
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MM
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40 41
MM
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AA
LL
PP
42
WW
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RR
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CC
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43
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44
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45
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47
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48
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49
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50
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51
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52
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53
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PP
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54
XX
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55
RR
CC
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MM
56 57 58 59 60 61 62
RREETT
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GG
JJ
63
VV
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HH
GG
RR
NN
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FF
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64 65
RR
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66
NN
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67
LL
PP
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68
RRLLXXPP
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69
PPTT
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70
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71
MM
AA
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72
RRLL
PP
MM
HH
FF
DD
73
LLAA
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74
MMRR
75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
HHQQ
NN
23
LL
EE
TT
4
RR
JJ
TTNN
WW
KK
MM
YY
5
LL
TT
FF
KK
II
6
NN
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7
RR
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89
10 11
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12
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13
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14 15 16 17
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TT
RR
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AA
18
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19 20 21
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A
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22
VV
MM
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RR
SS
HH
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YY
II
23
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24
RR
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25
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26SS
27
JJXX
28
LL
DD
29 30
SS
RR
KK
JJ
WW
GG
XX
31
AA
VV
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YY
TT
GG
PP
LL
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NN
HH
32
LLGG
FF
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AA
33 34
R
S
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KK
CC
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WW
35
NN
RR
VV
AA
GG
LL
CC
HH
TT
PP
36 37
SSPP
GG
LL
RR
TT
VV
AA
KK
EE
38
KK
HH
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EE
YY
NN
39
SS
RR
TT
XX
MM
KK
NN
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II
40 41
RR
AA
PP
LL
VV
HH
42
AA
EE
RR
NN
TT
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LL
SS
CC
HH
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YY
43
RR
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44
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PP
LL
GG
45
GG
LL
TT
VV
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WW
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CC
46
LL
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RR
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MM
KK
CC
NN
47
JJ
HH
EE
GG
KK
MM
CC
NN
LL
YY
SS
48
JJ
LL
PP
RR
VV
AA
GG
FF
WW
49
JJ
SS
EE
LL
CC
HH
YY
50 51
RR
TT
EE
NN
GG
DD
LL
52 53
RR
JJ
FF
GG
LL
HH
TT
PP
YY
NN
KK
54
PP
RR
JJ
VV
XX
II
YY
55 56 57 58 59 60 61 62
LLTT
JJ
GG
63 64EE
65
RR
AA
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CC
66
RR
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KK
PP
JJ
WW
NN
67
GG
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WW
RR
SS
XX
68
FF
RR
EE
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MM
PP
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69
LL
TT
SS
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70
RR
SS
AA
JJ
FF
LL
VV
YY
MM
NN
II
71
AA
GG
RR
TT
LL
PP
HH
SS
72
SS
TT
RR
PP
FF
HH
73
RR
JJ
VV
SS
AA
74RR
75
RR
LL
WWEE
76
QQTT
CC
GG 0
1
2
3
4
bits
1
GG
TT
FF
JJ
MM
HH
NN
2
RRFF
HH
CC
3
GG
VV
LL
TT
EE
4
JJ
WW
NN
MM
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YY
5
VV
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6
FF
VV
MM
WW
EE
NN
LL
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PP
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7
GG
AA
KK
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89
10
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12
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13
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14 15 16
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SS
17
SS
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MM
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LL
WW
II
PP
AA
18
VVPP
XX
19 20 21 22
TT
CC
EE
VV
SSDD
HH
YY
RR
MM
II
JJ
23
DD
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EE
24
RR
VV
LL
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YY
25
SS
FF
NN
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26
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27
VV
EE
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28
RR
GG
VV
KK
AA
29
VV
LL
30
LL
TT
SS
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XX
31
WW
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AA
PP
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NN
32
LL
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YY
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PP
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33
VV
FF
34
RR
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35
FF
RR
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EE
AA
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36 37
SS
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38
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39
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40 41
GG
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42
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43
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44
TT
KK
JJ
MM
RR
WW
PP
VV
HH
SS
LL
GG
45
LL
SS
GG
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II
46
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SS
KK
CC
NN
47
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RR
GG
VV
WW
EE
HH
LL
YY
NN
KK
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CC
48
SS
LL
RR
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VV
AA
GG
WW
FF
JJ
49
SS
RR
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YY
HH
50 51
SS
VV
RR
TT
WW
EE
GG
YY
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LL
52
VVKK
53
SS
VV
RR
MM
WW
CC
NN
TT
PP
LL
GG
HH
KK
54
RR
NN
VV
XX
YY
II
55 56 57 58 59 60 61 62
LL
KK
RR
TT
HH
YY
NN
GG
FF
63 64
AAEE
65
SS
GG
NN
YY
LL
MM
TT
CC
FF
66
LLGG
FF
SS
VV
RR
MM
YY
TT
KK
PP
NN
JJ
WW
67
GG
VV
LL
SS
RR
XX
68
RR
LL
SS
JJ
MM
FF
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PP
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EE
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AA
69
SS
WW
VV
GG
EE
AA
CC
FF
70
TT
KK
GG
JJ
AA
RR
LL
DD
VV
MM
NN
II
71
FF
JJ
GG
MM
RR
WW
CC
AA
VV
TT
LL
HH
PP
SS
72
GG
TTJJ
MM
RR
PP
HH
FF
DD
73
RR
MM
VV
JJ
SS
AA
74
WW
RR
75
RR
WW
LL
YYEE
76
VV
EE
WW
QQGG
TT
CC 0
1
2
3
4
bits
1
JJ
DDQQ
NN
2HH
3
GG
VV
EE
LL
MM
DD
JJ
TT
4
AA
FF
RR
DD
CC
HH
WW
NN
KK
YY
5
AA
KK
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FF
HH
EE
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WW
RR
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II
6
VV
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MM
NN
LL
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CC
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TT
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RR
GG
PP
7
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KK
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AA
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II
89
10 11
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12
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13
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14 15 16
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XX
NN
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CC
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GG
RR
SS
WW
17
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KK
EE
TT
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LL
DD
VV
PP
FF
WW
AA
II
18
RRJJ
PP
XX
19 20
DD
RR
21 22
GG
KK
EE
CC
VV
SS
MM
RR
HH
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II
YY
23
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24
SS
LL
25
SS
FF
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26
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27
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JJ
XX
28
LLGG
FF
AA
VV
KK
29
LL
GG
30
LL
EE
PP
WW
SS
RR
AA
TT
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XX
31
PP
RR
EE
TT
JJ
FF
YY
SS
AA
LL
VV
HH
KK
NN
32
SS
YY
CC
LL
RR
FF
VV
AA
TT
PP
GG
EE
33
SS
LL
MM
34
GG
RR
YY
LL
SS
TT
EE
PP
FF
HH
CC
MM
WW
35
SS
RR
EE
AA
LL
GG
HH
VV
TT
PP
36 37
SS
RR
TT
GG
LL
VV
AA
KK
PP
EE
38
KK
YY
NN
EE
HH
DD
39
SS
RR
XX
MM
KK
NN
JJ
TT
II
40 41
RR
AA
PP
VV
LL
HH
42
GG
RR
EE
HH
AA
TT
KK
NN
JJ
LL
FF
SS
YY
MM
43
AA
LL
HH
44
MM
JJ
AA
WW
RR
PP
SS
GG
CC
VV
LL
45
PP
EE
GG
LL
MM
YY
SS
JJ
FF
HH
WW
CC
II
46
EE
GG
VV
LL
TT
SS
RR
NN
JJ
KK
CC
MM
47
VV
MM
HH
WW
GG
NN
CC
LL
YY
SS
KK
48
AA
LL
KK
HH
PP
VV
GG
RR
FF
WW
49
KK
MM
LL
CC
JJ
YY
HH
50
S
LL
CC
EE
JJ
AA
GG
MM
NN
KK
PP
TT
RR
FF
WW
HH
II
VV
XX
DD
51
SS
RR
TT
VV
AA
NN
HH
WW
EE
MM
GG
JJ
KK
DD
LL
52
EE
PP
53
LLAA
EE
GG
RR
YY
CC
TT
II
FF
JJ
NN
KK
MM
HH
PP
54
LL
PP
KK
RR
CC
NN
VV
JJ
XX
YY
II
55 56 57 58 59 60 61 62
SS
TT
LL
KK
RR
PP
NN
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MM
HH
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63 64
AAEE
65
SS
RR
KK
EE
NN
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AA
MM
TT
GG
LL
FF
CC
66
SS
VV
TT
JJ
RR
LL
NN
PP
WW
67
PP
WW
JJ
RR
VV
GG
LL
SS
68
SS
KK
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II
CC
LL
JJ
EE
FF
PP
VV
GG
AA
TT
HH
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XX
69
GGKK
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WW
CC
TT
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VV
MM
EE
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AA
YY
70
WW
CC
AA
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RR
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II
71
KK
AA
CC
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RR
GG
LL
HH
PP
SS
72
EE
GG
RR
JJ
PP
DD
HH
FF
MM
73
PP
JJ
WW
VV
AA
SS
74
VVRR
75
RR
VV
LLMM
EE
76
SS
KKTT
GG
CC 0
1
2
3
4
bits
1NN
23
NNTT
CC
4
RRMM
NN
WW
KK
YY
5
KK
VV
RR
LL
EEII
6
VV
LLRR
DD
GG
PP
AA
TT
7
KK
AA
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WW
89
10
SS
YY
11
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LL
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WW
EE
12
JJ
SSVV
TT
RR
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GG
FF
AA
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MM
KK
NN
13
RR
GG
EE
14 15 16
PPKK
NN
MM
HH
AA
VV
RR
JJ
LL
SS
CC
YY
DD
TT
GG
FF
WW
17
RR
NN
VV
LL
TT
WW
DD
AA
FF
II
PP
18
RRJJPP
XX
19 20 21
DD
NN
LL
AA
VV
TT
FF
GG
XX
CC
WW
HH
KK
MM
YY
PP
II
SS
JJ
22
AA
VV
SS
JJ
RR
DD
II
HH
YY
23
SS
RR
EE
24
GG
LLJJ
25
RRSS
26
RR
GGSS
27
JJXX
28
GG
SS
DD
VV
29
RRLL
MM
GG
30
SS
RR
TT
JJ
HH
PP
KK
GG
WW
XX
31
DD
RR
TT
VV
GG
PP
LL
AA
KK
NN
32
LLKK
SS
RR
TT
GG
AA
PP
VV
EE
33
VV
SS
34
TT
LL
GG
PP
SS
CC
WW
35
SSRR
MM
LL
AA
TT
GG
VV
HH
PP
36 37
SS
RR
GG
TT
PP
VV
LL
AA
EE
KK
38
HH
YY
DD
KK
NN
EE
39
SS
RR
II
XX
MM
NN
TT
KK
JJ
40 41
VV
RR
LL
AA
PP
HH
42
RR
KK
SS
AA
NN
TT
LL
JJ
FF
HH
YY
43
PP
RRJJ
44
RR
TT
SS
PP
LL
VV
JJ
GG
45
AA
GG
PP
SS
WW
LL
EE
CC
HH
FF
II
46
TT
GG
JJ
CC
YY
LL
RR
SS
NN
KK
MM
47
RR
GG
HH
CC
MM
SS
LL
NN
KK
YY
48
KK
GG
VV
RR
JJ
FF
WW
49
RR
LL
KKJJ
HH
CC
YY
50
LL
KK
JJ
CCVV
RR
PP
GG
FF
DD
AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
JJ
GG
DD
LL
52 53
RR
KK
LL
NN
PP
TT
GG
SS
HH
YY
54
RR
NN
JJ
VV
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
RRTT
KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
FF
EE
70
RR
VV
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
function logos
Genome
g
tRNA
genes
T
g
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g tRNA CIFs are Nonetheless Highly
Evolutionarily Conserved tRNA Class Informative Features
(CIFs) are Based on Physics, not
Evolutionary Conservation
Rare bases carry more functional information
in Drosophila tRNA genes
Phillips and Ardell (2021). J. Mol. Evol. 89:103
in Drosophila tRNA genes
Phillips and Ardell (2021). J. Mol. Evol. 89:103
Non-Watson-Crick Base Pairs are Conserved
Class-Informative Features in Trypanosome tRNAs
aminoacylationsignal.Thesefourcandidateswerethenre-screenedusingtime-dependent
quantitativeapproachesandweconcludedthatthreeofthefourmixes,1881C,2059D,and
2096Bwerealteringaminoacylation,withinhibitoryactivitiesrangingbetween80%and99%
(Fig9C).Usingmeansandstandarddeviationsoffourreplicatescintillationcount-per-minute
endpointsundertheDMSOcontrolconditionorwithoutaddedenzyme,wecalculatedan
acceptableZ-factorof0.67forthisfollow-upaminoacylationtime-course-basedassay(S6
Table).
Fig7.FunctionlogosfortRNAClass-InformativeBase-PairsandClass-InformativeMis-PairsintheL.majorclade.Themeaningsofletters,stackheightsandletter
heightsareallthesameasinFig5.
https://doi.org/10.1371/journal.pntd.0007983.g007
TargetingtRNA-synthetaseinteractionsineukaryoticpathogens
PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0007983February27,2020 16/30
SincetheaminoacylationscreendiscernstotalnetchangestotheaaRSactivity,we
attemptedtoidentifywhichstepoftheaaRScatalyzedreactionisbeingaffectedbytheMNPs.
ToobserveanytRNA-independenteffectsonaaRSfunction,weusedpyrophosphateexchange
tomonitorATP-dependentaminoacidactivation.Fromthisexperiment,wewereabletocon-
cludethatourinhibitorswereperturbingaminoacidactivation,withleadcompoundsranging
ininhibitoryactivitybetween45%and95%.ThedifferencesinMNPactivitybetweenamino
acidactivationandtRNA-dependentaminoacylationhighlightthemultipleaaRSactivities
thatcanbetargetedinournetworkpredictions.Tovalidatethepredictivetoolforidentifying
anti-trypanosomaldrugs,wecounter-screenedthenewlyidentifiedLm.AlaRSinhibitors
Fig8.FunctionlogosfortRNAClass-InformativeBase-PairsandClass-InformativeMis-PairsintheAmericanTrypanosomaclade.Themeaningsofletters,stack
heightsandletterheightsareallthesameasinFig5.
https://doi.org/10.1371/journal.pntd.0007983.g008
TargetingtRNA-synthetaseinteractionsineukaryoticpathogens
PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0007983February27,2020 17/30
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
Class-Informative Features in Trypanosome tRNAs
aminoacylationsignal.Thesefourcandidateswerethenre-screenedusingtime-dependent
quantitativeapproachesandweconcludedthatthreeofthefourmixes,1881C,2059D,and
2096Bwerealteringaminoacylation,withinhibitoryactivitiesrangingbetween80%and99%
(Fig9C).Usingmeansandstandarddeviationsoffourreplicatescintillationcount-per-minute
endpointsundertheDMSOcontrolconditionorwithoutaddedenzyme,wecalculatedan
acceptableZ-factorof0.67forthisfollow-upaminoacylationtime-course-basedassay(S6
Table).
Fig7.FunctionlogosfortRNAClass-InformativeBase-PairsandClass-InformativeMis-PairsintheL.majorclade.Themeaningsofletters,stackheightsandletter
heightsareallthesameasinFig5.
https://doi.org/10.1371/journal.pntd.0007983.g007
TargetingtRNA-synthetaseinteractionsineukaryoticpathogens
PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0007983February27,2020 16/30
SincetheaminoacylationscreendiscernstotalnetchangestotheaaRSactivity,we
attemptedtoidentifywhichstepoftheaaRScatalyzedreactionisbeingaffectedbytheMNPs.
ToobserveanytRNA-independenteffectsonaaRSfunction,weusedpyrophosphateexchange
tomonitorATP-dependentaminoacidactivation.Fromthisexperiment,wewereabletocon-
cludethatourinhibitorswereperturbingaminoacidactivation,withleadcompoundsranging
ininhibitoryactivitybetween45%and95%.ThedifferencesinMNPactivitybetweenamino
acidactivationandtRNA-dependentaminoacylationhighlightthemultipleaaRSactivities
thatcanbetargetedinournetworkpredictions.Tovalidatethepredictivetoolforidentifying
anti-trypanosomaldrugs,wecounter-screenedthenewlyidentifiedLm.AlaRSinhibitors
Fig8.FunctionlogosfortRNAClass-InformativeBase-PairsandClass-InformativeMis-PairsintheAmericanTrypanosomaclade.Themeaningsofletters,stack
heightsandletterheightsareallthesameasinFig5.
https://doi.org/10.1371/journal.pntd.0007983.g008
TargetingtRNA-synthetaseinteractionsineukaryoticpathogens
PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0007983February27,2020 17/30
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
109Journal of Molecular Evolution (2021) 89:103–116
1 3
Figure!4 shows that a great deal of functional informa-
tion resides in Base-Pairs and Base Mis-Pairs of Drosophila
melanogaster tRNAs. Several base mis-pairs are function-
ally informative, including U13:U22 in the D-arm, which
is associated to both tRNA
Pro
and tRNA
Val
(contained in 32
tRNA genes of 32 total), U31:U39 in the C-arm contained
in all six tRNA
Met
genes, C50:U64 in the T-arm contained in
nine of 11 tRNA
Ile
genes, G13:A22 in the D-arm contained
in all 42 tRNA
Ser
and tRNA
Leu
genes, and a G4:G69 mis-
pair in the acceptor stem associated with nine of 16 tRNA
Thr
genes. We show a plot of the significance of paired-site CIFs
as a function of CIF information in Supplementary Fig. S3.
A text-file containing all statistics for paired-site CIFs in
D. melanogaster, including frequencies of CIFs in genes of
various functions, as well as p-values and FDRs, is provided
in the Supplementary Code and Data.
Fig. 4 Class-Informative Base-Pairs and Base Mis-Pairs from 288
re-annotated tRNA genes in Drosophila melanogaster with COVE
scores of at least 50 bits as computed in tSFM v1.0.1 with a Benja-
mini–Hochberg False Discovery Rate of 5% (CIFs not meeting this
significance threshold are not shown). The total height of a stack of
letters quantifies information gained about the functional type of a
tRNA by a tRNA-binding protein if it specifically recognizes the
paired features indicated. The letters within each stack symbolize
functional types of tRNA genes, wherein IUPAC one-letter amino
acid codes represent elongator tRNA aminoacylation identities and
“X” symbolizes initiator tRNAs. The relative heights of letters within
each stack quantify the over-representation of tRNA functional types
carrying that feature relative to the background frequency determined
by the frequencies of genes of various functional types (as calculated
through normalized log-odds)
Non-Watson-Crick Base Pairs are also
Informative Features in Drosophila tRNAs
Phillips and Ardell (2021). J. Mol. Evol. 89:103
1 3
Figure!4 shows that a great deal of functional informa-
tion resides in Base-Pairs and Base Mis-Pairs of Drosophila
melanogaster tRNAs. Several base mis-pairs are function-
ally informative, including U13:U22 in the D-arm, which
is associated to both tRNA
Pro
and tRNA
Val
(contained in 32
tRNA genes of 32 total), U31:U39 in the C-arm contained
in all six tRNA
Met
genes, C50:U64 in the T-arm contained in
nine of 11 tRNA
Ile
genes, G13:A22 in the D-arm contained
in all 42 tRNA
Ser
and tRNA
Leu
genes, and a G4:G69 mis-
pair in the acceptor stem associated with nine of 16 tRNA
Thr
genes. We show a plot of the significance of paired-site CIFs
as a function of CIF information in Supplementary Fig. S3.
A text-file containing all statistics for paired-site CIFs in
D. melanogaster, including frequencies of CIFs in genes of
various functions, as well as p-values and FDRs, is provided
in the Supplementary Code and Data.
Fig. 4 Class-Informative Base-Pairs and Base Mis-Pairs from 288
re-annotated tRNA genes in Drosophila melanogaster with COVE
scores of at least 50 bits as computed in tSFM v1.0.1 with a Benja-
mini–Hochberg False Discovery Rate of 5% (CIFs not meeting this
significance threshold are not shown). The total height of a stack of
letters quantifies information gained about the functional type of a
tRNA by a tRNA-binding protein if it specifically recognizes the
paired features indicated. The letters within each stack symbolize
functional types of tRNA genes, wherein IUPAC one-letter amino
acid codes represent elongator tRNA aminoacylation identities and
“X” symbolizes initiator tRNAs. The relative heights of letters within
each stack quantify the over-representation of tRNA functional types
carrying that feature relative to the background frequency determined
by the frequencies of genes of various functional types (as calculated
through normalized log-odds)
Non-Watson-Crick Base Pairs are also
Informative Features in Drosophila tRNAs
Phillips and Ardell (2021). J. Mol. Evol. 89:103
Phillips and Ardell (2021). J. Mol. Evol. 89:103
(Batey et al+,2000)+As shown in the right panel of
Figure 9,this symmetric internal loop is very similar to
the submotifs of the bacterial loop E motif (Fig+9,left
panel)+The SRP motif comprises atransHoogsteen/
Sugar-edge A•C pair adjoining atransW+C+/Hoogsteen
C•A pair followed by atransbifurcated G•G pair and a
cisW+C+/W+C+A•G pair+The trans Hoogsteen/Sugar-
edge A•C pair corresponds to the A•G pair in the loop
E submotif and is isosteric to it (Fig+10,left)+Thetrans
W+C+/Hoogsteen C•A pair corresponds to the U•A pair
in loop E (Fig+10,right)+ThecisW+C+/W+C+A•G corre-
sponds to the water-inserted A•G in the loop E motif,
which is alsocisW+C+/W+C+,with an H-bond between
AN6 and GO6 and the water molecule bridging the
imino nitrogens+The bifurcated G•G in the SRP differs
slightly from the pair in loop E,as shown above in
Figure 8+The loop E submotif occurs also in helix 20 of
16S rRNA (Wimberly et al+,2000),as was predicted
(Leontis & Westhof,1998a)+Interestingly,the G•G bi-
furcated pair in 16S rRNA is identical to the pair in the
SRP loop (transbifurcated as in Fig+8B)+
Recognition of motif similarity in annotated
three-dimensional structures
Because the classification facilitates the comparison
between different three-dimensional structures to iden-
tify common three-dimensional motifs,it further aids in
predicting families of isosteric pairings that can substi-
tute for each other in homologous RNA molecules+Since
three-dimensional structures of homologous RNA mol-
ecules are more strongly conserved than their individ-
ual sequences,covariation data can be used to identify
bases involved in tertiary interactions and even indi-
cate the most likely pairing geometry+This approach
was successfully applied for predicting potential sarcin-
ricin motifs (also frequently referred to as “S-turn” or
“eukaryal 5S loop E” motifs) and bacterial loop E motifs
in 16S and 23S rRNAs (Leontis & Westhof,1998a,
1998b)+All these motifs,except for one,were later iden-
tified in crystal structures of the ribosome 70S and its
subunits (Cate et al+,1999;Nissen et al+,2000;Schlu-
enzen et al+,2000;Wimberly et al+,2000)+In ad-
FIGURE 10.Comparison of isosteric base pairs in bacterial loop E (URL064) and the internal loop of Domain IV SRP 4+5S
RNA (Correll et al+,1997;Batey et al+,2000)+Left panel:transHoogsteen/Sugar edge pairs A104•G72 from loop E and
A164•C147 from 4+5 S RNA+Right panel:transWatson–Crick/Hoogsteen pairs U103•A73 from loop E and C163•A148 from
4+5 S RNA+
510 N.B. Leontis and E. Westhof
Cold Spring Harbor Laboratory Press on June 13, 2022 - Published by rnajournal.cshlp.orgDownloaded from
Figure 9,this symmetric internal loop is very similar to
the submotifs of the bacterial loop E motif (Fig+9,left
panel)+The SRP motif comprises atransHoogsteen/
Sugar-edge A•C pair adjoining atransW+C+/Hoogsteen
C•A pair followed by atransbifurcated G•G pair and a
cisW+C+/W+C+A•G pair+The trans Hoogsteen/Sugar-
edge A•C pair corresponds to the A•G pair in the loop
E submotif and is isosteric to it (Fig+10,left)+Thetrans
W+C+/Hoogsteen C•A pair corresponds to the U•A pair
in loop E (Fig+10,right)+ThecisW+C+/W+C+A•G corre-
sponds to the water-inserted A•G in the loop E motif,
which is alsocisW+C+/W+C+,with an H-bond between
AN6 and GO6 and the water molecule bridging the
imino nitrogens+The bifurcated G•G in the SRP differs
slightly from the pair in loop E,as shown above in
Figure 8+The loop E submotif occurs also in helix 20 of
16S rRNA (Wimberly et al+,2000),as was predicted
(Leontis & Westhof,1998a)+Interestingly,the G•G bi-
furcated pair in 16S rRNA is identical to the pair in the
SRP loop (transbifurcated as in Fig+8B)+
Recognition of motif similarity in annotated
three-dimensional structures
Because the classification facilitates the comparison
between different three-dimensional structures to iden-
tify common three-dimensional motifs,it further aids in
predicting families of isosteric pairings that can substi-
tute for each other in homologous RNA molecules+Since
three-dimensional structures of homologous RNA mol-
ecules are more strongly conserved than their individ-
ual sequences,covariation data can be used to identify
bases involved in tertiary interactions and even indi-
cate the most likely pairing geometry+This approach
was successfully applied for predicting potential sarcin-
ricin motifs (also frequently referred to as “S-turn” or
“eukaryal 5S loop E” motifs) and bacterial loop E motifs
in 16S and 23S rRNAs (Leontis & Westhof,1998a,
1998b)+All these motifs,except for one,were later iden-
tified in crystal structures of the ribosome 70S and its
subunits (Cate et al+,1999;Nissen et al+,2000;Schlu-
enzen et al+,2000;Wimberly et al+,2000)+In ad-
FIGURE 10.Comparison of isosteric base pairs in bacterial loop E (URL064) and the internal loop of Domain IV SRP 4+5S
RNA (Correll et al+,1997;Batey et al+,2000)+Left panel:transHoogsteen/Sugar edge pairs A104•G72 from loop E and
A164•C147 from 4+5 S RNA+Right panel:transWatson–Crick/Hoogsteen pairs U103•A73 from loop E and C163•A148 from
4+5 S RNA+
510 N.B. Leontis and E. Westhof
Cold Spring Harbor Laboratory Press on June 13, 2022 - Published by rnajournal.cshlp.orgDownloaded from
Outline
tRNA CIFs Predict Functional
Divergences Between Human and
Trypanosome aaRSs Rare tRNA CIFs are More
Functionally Informative scores Cyano-MLP
13
0.0
1.0
classification
probabilities0
1
2
3
4
bits
1
AA
SSMM
XX
JJ
FFNN
QQ
2
GG
JJ
CC
WW
YY
HH
3
GG
NN
VV
LL
CC
TT
EE
4
TT
JJ
EE
VV
AA
CC
RR
WW
HH
NN
MM
KK
YY
5
LL
GGKK
QQ
TT
JJ
FF
NN
AA
RR
YY
WW
II
6
QQ
VV
EE
MM
YY
JJ
KK
NN
HH
LL
RR
GG
AA
PP
DD
TT
7
SS
RR
GGNN
YY
KK
AA
VV
WW
JJ
II
89
GG
JJ
FF
10
SS
RRYY
11
RR
AA
NN
LL
TT
PP
QQ
YY
HHDD
EE
GG
WW
12
PP
JJSSWW
KK
VV
GG
MM
TT
NN
RR
AA
II
FF
DD
13
KK
YY
JJ
LL
CC
TT
AA
RR
QQ
MM
WW
GG
EE
14
SS
AA
YY
15
KK
QQJJ
16
PP
II
EE
KK
XX
HH
QQ
RR
SSNN
JJ
DD
WW
YY
VV
AA
TT
GG
FF
CC
MM
LL
17
SS
YY
HH
EE
QQ
NN
KK
TT
VV
RR
DD
FF
LL
WW
PP
AA
II
18
SS
TT
EEXX
PP
19 20 21
EE
JJ
YYLLDD
WW
KK
AA
TT
SS
MM
QQ
HH
XX
NN
FF
GG
PP
II
CC
VV
22
GG
PP
FFWW
NN
XX
CC
KK
EE
JJ
VV
SS
MM
HH
YY
DD
RR
II
23
PP
DD
NN
SS
RREE
24
YY
MM
JJ
LL
KK
25
KK
NN
FF
SS
QQYY
26
TTYY
KK
SS
27
MM
QQ
YYXX
28
SS
QQ
JJ
FF
EE
MM
VV
LL
AA
YY
TT
WW
KK
GG
RR
29
RR
LL
MM
NN
GG
WW
30
PP
VV
SS
LL
QQ
YY
JJ
FF
EE
WW
AA
MM
NNKK
TT
RR
CC
GG
XX
31
RRYY
WW
QQ
JJ
CC
EE
PP
TT
LL
DD
SS
GG
VV
FF
KK
NN
AA
HH
32
GGJJ
YY
DD
LL
MM
KK
SS
FF
TT
RR
AA
QQ
VV
PP
EE
33
AASS
34
VV
SS
JJ
EE
AA
MM
NN
TT
RR
DD
LL
GG
YY
XX
PP
FF
QQ
HH
CC
WW
35
KK
YYNN
FF
EE
MM
SS
RR
AA
GG
LL
QQ
VV
TT
HH
PP
36 37
SS
LL
TT
GG
PP
VV
RR
AA
KK
QQ
EE
38
NN
QQ
EE
HH
DD
YY
KK
39
SS
RR
KK
TT
XX
MM
II
JJ
NN
40AA
41
SSGG
RR
AA
PP
LL
VV
QQ
HH
42
NN
GG
QQ
EE
RR
AA
KK
HH
TT
YY
FF
LL
SS
JJ
43
TT
GG
LL
EE
DD
NN
AA
QQ
SS
MM
RR
YY
CC
JJ
44
FF
AA
NN
HH
JJ
KK
XX
MM
YY
RR
PP
VV
SS
LL
GG
45
KK
GG
PP
DD
MM
HH
RR
SS
EE
LL
YY
TT
FF
CC
WW
II
46
QQ
JJ
FF
MM
GG
YY
RR
WW
TT
CC
LL
SS
KK
NN
47
AAQQ
FF
DDTT
WW
RR
JJ
EE
GG
MM
HH
CC
LL
YY
KK
NN
SS
48
TT
KK
HH
SS
NN
AA
JJ
LL
CC
MM
PP
VV
GG
RR
FF
WW
49
RR
KK
FF
EE
SS
JJ
CC
HH
LL
QQ
YY
50
YY
JJ
SS
LL
EE
CC
GGQQMM
WW
AA
KK
RR
FF
TT
NN
VV
PP
XX
II
DD
HH
51
SS
PP
FF
CC
HH
JJ
VV
YY
TT
AA
NN
RR
MM
WW
EE
GG
QQ
KK
DD
LL
52
GG
II
KK
53
VV
JJ
AA
CC
MM
LL
KK
GG
QQ
RR
WW
YY
TT
SS
FF
PP
HH
NN
54
QQ
TT
MM
LL
PP
NN
JJ
VV
XX
YY
II
55
GG
NN
YY
56 57 58 59YY
60 61 62
AA
RRDD
EE
CC
XX
KK
LL
QQ
MM
YY
FF
NN
TT
GG
HH
JJ
63
DD
QQ
FF
TT
YY
AA
HH
NN
RR
MM
EE
JJ
LL
KK
VV
GG
II
XX
SS
CC
PP
WW
64
VVYYEE
65
RR
VV
SS
NN
AA
GG
YY
XX
DD
WW
FF
TT
LL
CC
66
AA
SS
YY
FF
MM
XX
TT
LL
JJ
VV
RR
KK
PP
WW
NN
QQ
67
GG
MM
KK
YY
WW
LL
QQ
SS
RR
68
SS
NNMM
QQ
FF
KK
LL
JJ
GG
PP
TT
EE
XX
VV
AA
HH
DD
69
RRSS
JJ
TT
MMLL
KK
DD
NN
WW
YY
VV
GG
XX
AA
EE
FF
CC
70
KK
SS
GG
WW
NN
LL
RR
JJ
DD
QQ
AA
MM
FF
VV
II
71
YY
FF
NN
KK
JJ
WW
QQ
AA
CC
TT
GG
RR
LL
VV
HH
PP
SS
72
SS
YY
TT
GG
AA
NN
XXKK
JJ
RR
PP
MM
HH
FF
DD
73
RRTT
NN
JJ
WW
YY
VV
AA
SS
74
YY
DD
NN
XX
WW
RR
75
TT
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NN
VV
RR
MM
LL
YY
XX
EE
76
SS
KK
QQ
JJ
MM
EE
YYGG
TT
CC 0
1
2
3
4
bits
1
RR
PP
SS
LL
KK
EE
FF
MM
JJ
XXNN
QQ
2
SS
PPJJ
II
MM
WW
CC
HH
3
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II
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4
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5
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6
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7
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89
10
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11
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12
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13
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14 15
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16
II
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17
SS
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QQ
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MM
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II
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AA
18
TT
II
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XX
19 20 21
EE
LL
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22
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23
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24
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25
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26
VV
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27
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28
PP
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29
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30
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31
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32
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33
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34
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35
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VV
AA
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36 37
IISS
RR
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38
YY
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39
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40DD
41
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42
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43
II
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44
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45
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46
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47
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48
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49
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50
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51
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52
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53
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54
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55
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56
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57 58 59
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60 61 62
RR
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63
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YY
WW
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NN
PP
LL
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VV
MM
XX
II
64
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65
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YY
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66
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XX
TT
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NN
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67
AA
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68
II
QQ
RR
SS
NN
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GG
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XX
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69
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XX
70
PPHH
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II
71
GG
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72
LL
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73
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74
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75
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QQ
VV
WW
RR
JJ
MM
LLEE
76
RR
LLKK
FF
SS
XX
EETT
GG
CC 0
1
2
3
4
bits
1
AA
GG
VV
KKNN
QQ
23
LL
RR
VV
FF
EE
CC
TT
4
AA
GG
VV
LL
CC
DD
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5
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6
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7
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89
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12
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AA
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13
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14 15 16
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II
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17
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AA
18
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19 20 21
R
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NN
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II
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22
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PP
GG
EE
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II
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23
PP
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SSRREE
24
LL
MM
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25
TT
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QQ
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26
PP
GG
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MMSS
27
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28
SS
LL
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NN
GG
PP
WW
RR
TT
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AA
VV
29
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NN
GG
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LL
30
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CC
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WW
GG
XX
31
EE
XX
RR
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CC
FF
SS
VV
DD
TT
GG
AA
PP
LL
HH
KK
NN
32
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II
KK
NN
CC
YY
QQ
FF
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RR
TT
AA
PP
VV
EE
33
LL
AA
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34
GG
VV
II
RR
AA
JJ
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DD
FF
TT
LL
QQ
SS
HH
PP
CC
WW
35
II
KK
EE
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SS
CC
RR
AA
VV
GG
LL
TT
QQ
PP
HH
36 37
FF
IISS
RR
PP
TT
GG
LL
AA
VV
KK
EE
QQ
38
FFNN
EE
DD
QQ
KK
HH
YY
39
FFSS
RR
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40
RRDD
41
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LL
VV
QQ
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42
VV
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YY
43
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44
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45
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II
46
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47
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VV
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48
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49
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51
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52
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53
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LL
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RR
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54
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TT
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II
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XX
55
VV
MM
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56 57 58 59 60 61 62
II
AA
LL
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NN
RR
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63
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RR
HH
FF
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64
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65
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II
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AA
NN
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CC
66
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II
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NN
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67
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68
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NN
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HH
69
KK
DD
II
RR
WW
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XX
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70
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GG
XX
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AA
VV
NN
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71
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NN
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72
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73
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NN
WW
CC
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74
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II
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NN
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RR
75
PP
VV
NN
LL
MM
JJ
RR
WW
XXEE
76
RRKKGG
TT
CC 0
1
2
3
4
bits
1
AAMMNN
QQ
2
EEMM
3
WWRR
4
VV
EE
RRWWMM
KK
YY
NN
5
AAII
LL
RR
TT
6
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MM
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7
GG
SSKKRR
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AA
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VV
89
VV
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10 11
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12
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13
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14
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15 16
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17
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18XX
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19 20 21
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22
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23
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24
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25
VVSSYY
26
CC
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27
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28
LLGG
29SS
30
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31
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NN
32
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33
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34
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35
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PP
36 37
RR
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38
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NN
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39
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II
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XX
MM
JJ
NN
40 41
MM
VVRR
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AA
LL
PP
QQ
42
WW
HHVV
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SS
RR
NN
CC
JJ
YY
FF
43
LLQQ
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44
MMSS
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VV
GG
45
LL
MM
SS
WW
II
CC
46
LL
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CC
NN
47
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GG
HH
LL
SS
KK
YY
NN
CC
48
RR
PP
GG
VV
AA
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49
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YY
QQ
50
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LLSS
GG
PP
HH
TT
CC
VV
RR
AA
QQ
DD
NN
KK
XX
MM
FF
II
WW
51
EEGGDD
LL
52
RRTT
53
VVLL
RR
PP
TT
SS
KK
QQ
YY
54
XX
TT
RRVV
II
YY
55
RR
CC
WWTT
MM
56 57 58 59 60 61 62
RREETT
VV
GG
JJ
63
VV
DD
EE
HH
GG
QQ
RR
NN
KK
FF
MM
YY
II
CC
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LL
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64 65
RR
TT
AA
GG
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MM
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66
NN
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XX
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WW
QQ
67
LL
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68
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VV
GG
TT
HH
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69
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MM
AA
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70
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II
71
MM
AA
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TT
RR
PP
GG
LL
SS
72
RRLL
PP
MM
HH
FF
DD
73
LLAA
SS
74
MMRR
75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
HHQQ
NN
23
LL
EE
TT
4
RR
JJ
TTNN
WW
KK
MM
YY
5
LL
TT
FF
KK
II
6
NN
VV
MM
LL
PP
TT
RR
QQ
AA
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7
RR
KK
AA
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89
10 11
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NN
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12
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NN
13
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14 15 16 17
HH
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II
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AA
18
JJXX
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19 20 21
E
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A
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22
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23
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24
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25
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26SS
27
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JJXX
28
LL
QQ
DD
29 30
SS
QQ
RR
KK
JJ
WW
GG
XX
31
AA
VV
SS
YY
TT
GG
PP
LL
FF
KK
NN
HH
32
LLGG
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QQ
DD
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NN
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VV
AA
33 34
R
S
T
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DD
KK
CC
PP
WW
35
NN
RR
VV
QQ
AA
GG
LL
CC
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PP
36 37
SSPP
GG
LL
RR
TT
VV
AA
KK
QQ
EE
38
KK
HH
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NN
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39
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RR
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XX
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KK
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II
40 41
RR
AA
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LL
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42
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43
RR
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44
VV
RR
SS
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LL
GG
45
GG
LL
TT
VV
EE
SS
WW
FF
II
CC
46
LL
JJ
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SS
MM
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NN
47
JJ
HH
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GG
KK
MM
CC
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LL
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48
JJ
LL
PP
RR
VV
AA
GG
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49
JJ
SS
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LL
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QQ
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50 51
RR
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NN
GG
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52 53
RR
JJ
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GG
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54
PP
RR
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VV
XX
II
YY
55 56 57 58 59 60 61 62
LLTT
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GG
63 64EE
65
RR
AA
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66
RR
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67
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XX
68
FF
RR
EE
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AA
69
LL
TT
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AA
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CC
70
RR
SS
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71
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GG
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72
SS
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RR
PP
FF
HH
73
RR
JJ
VV
SS
AA
74RR
75
RR
LL
WWEE
76
QQTT
CC
GG 0
1
2
3
4
bits
1
GG
TT
FF
JJ
MM
HH
QQ
NN
2
RRFF
HH
CC
3
GG
VV
QQ
LL
TT
EE
4
JJ
WW
NN
MM
KK
YY
5
VV
TT
EE
KK
WW
RRII
6
FF
VV
MM
WW
EE
NN
LL
QQ
GG
RR
AA
PP
TT
DD
7
GG
AA
KK
VV
WW
JJ
II
89
10
HH
11
RR
LL
QQ
GG
EE
DD
WW
12
SS
JJ
WW
VV
RR
KK
AA
GG
TT
II
MM
FF
DD
NN
13
QQ
RR
WW
YY
GG
EE
14 15 16
PPKK
JJEE
QQ
VV
RR
DD
HH
CC
FF
XX
GG
NN
AA
TT
LL
WW
MM
YY
SS
17
SS
JJ
RR
KK
QQ
TT
FF
MM
VV
DD
LL
WW
II
PP
AA
18
VVPP
XX
19 20 21 22
TT
CC
EE
VV
SSDD
HH
YY
RR
MM
II
JJ
23
DD
RR
EE
24
RR
VV
LL
KK
YY
25
SS
FF
NN
YY
26
KKSS
27
VV
EE
WW
QQXX
28
RR
GG
VV
KK
QQ
AA
29
VV
LL
QQ
30
LL
TT
SS
PP
VV
AA
JJ
MM
QQ
CC
RR
WW
GG
XX
31
WW
GG
SS
RR
DD
AA
PP
EE
VV
TT
FF
LL
KK
HH
NN
32
LL
WW
SS
YY
FF
RR
TT
QQ
VV
AA
PP
EE
33
VV
FF
34
RR
GG
VV
TT
QQ
FF
PP
LL
CC
WW
35
FF
RR
SS
EE
AA
QQ
GG
LL
VV
TT
PP
HH
36 37
SS
GG
RR
PP
VV
LL
AA
TT
KK
QQ
EE
38
DD
EE
HH
KK
YY
NN
QQ
39
SS
RR
II
KK
JJ
TT
XX
NN
MM
40 41
GG
RR
PP
VV
AA
LL
QQ
HH
42
VV
KK
EE
AA
MM
QQ
NN
RR
LL
TT
FF
HH
YY
JJ
SS
43
GG
RR
CC
JJ
44
TT
KK
JJ
MM
RR
WW
PP
VV
HH
SS
LL
GG
45
LL
SS
GG
TT
WW
FF
CC
II
46
GG
WW
LL
TT
YY
RR
SS
KK
CC
NN
47
TT
RR
GG
VV
WW
EE
HH
LL
YY
NN
KK
SS
CC
48
SS
LL
RR
PP
VV
AA
GG
WW
FF
JJ
49
SS
RR
KK
LL
CC
JJ
QQ
YY
HH
50 51
SS
VV
RR
TT
QQ
WW
EE
GG
YY
KK
DD
LL
52
VVKK
53
SS
VV
RR
MM
WW
CC
NN
TT
PP
LL
QQ
GG
HH
KK
54
RR
NN
VV
XX
YY
II
55 56 57 58 59 60 61 62
LL
KK
RR
TT
HH
YY
NN
GG
FF
63 64
AAEE
65
SS
GG
NN
YY
LL
MM
TT
CC
FF
66
LLGG
FF
SS
VV
RR
MM
YY
TT
KK
PP
NN
JJ
WW
QQ
67
GG
VV
LL
QQ
SS
RR
XX
68
RR
LL
SS
JJ
MM
FF
GG
PP
VV
EE
TT
DD
HH
AA
69
SS
WW
VV
GG
EE
AA
CC
FF
70
TT
KK
GG
JJ
AA
QQ
RR
LL
DD
VV
MM
NN
II
71
FF
JJ
GG
MM
RR
WW
CC
AA
VV
TT
LL
HH
PP
SS
72
GG
TTJJ
MM
RR
PP
HH
FF
DD
73
RR
MM
VV
JJ
SS
AA
74
WW
RR
75
RR
WW
LL
YYEE
76
VV
EE
WW
QQGG
TT
CC 0
1
2
3
4
bits
1
JJ
DDQQ
NN
2HH
3
GG
VV
EE
LL
MM
DD
JJ
TT
4
AA
FF
RR
DD
CC
HH
WW
NN
KK
YY
5
AA
KK
LL
FF
HH
EE
TT
NN
WW
RR
DD
II
6
VV
KK
MM
NN
LL
QQ
EE
HH
CC
DD
TT
AA
RR
GG
PP
7
SS
KK
EE
AA
MM
JJ
VV
WW
II
89
10 11
RR
PP
LL
HH
GG
DD
EE
WW
12
PP
SS
KK
JJ
TT
AA
GG
WW
VV
RR
II
MM
NN
FF
DD
13
LL
SS
AAWW
DD
EE
GG
14 15 16
PP
II
EE
MM
KK
TT
XX
NN
QQ
DDHH
YY
CC
VV
AA
FF
JJ
LL
GG
RR
SS
WW
17
SS
KK
EE
TT
QQMM
RR
LL
DD
VV
PP
FF
WW
AA
II
18
RRJJ
PP
XX
19 20
DD
RR
21 22
GG
KK
EE
CC
VV
SS
MM
RR
HH
DD
II
YY
23
SSRR
DD
CCEE
24
SS
LL
25
SS
FF
NNYY
26
KKTT
SS
27
QQ
EE
JJ
XX
28
LLGG
FF
AA
VV
KK
29
LL
GG
30
LL
EE
PP
QQ
WW
SS
RR
AA
TT
JJ
VV
KK
GG
XX
31
PP
RR
EE
TT
JJ
FF
YY
SS
AA
LL
VV
HH
KK
NN
32
SS
YY
CC
LL
RR
FF
QQ
VV
AA
TT
PP
GG
EE
33
SS
LL
MM
34
GG
RR
QQ
YY
LL
SS
TT
EE
PP
FF
HH
CC
MM
WW
35
SS
RR
EE
AA
LL
GG
QQ
HH
VV
TT
PP
36 37
SS
RR
TT
GG
LL
VV
AA
KK
PP
QQ
EE
38
KK
YY
NN
EE
QQ
HH
DD
39
SS
RR
XX
MM
KK
NN
JJ
TT
II
40 41
RR
AA
PP
VV
LL
QQ
HH
42
GG
RR
EE
HH
AA
TT
KK
NN
JJ
LL
FF
SS
YY
MM
43
AA
LL
HH
44
QQ
MM
JJ
AA
WW
RR
PP
SS
GG
CC
VV
LL
45
PP
EE
GG
LL
MM
YY
SS
JJ
FF
HH
WW
CC
II
46
EE
GG
VV
LL
TT
SS
RR
NN
JJ
KK
CC
MM
47
VV
MM
HH
WW
QQ
GG
NN
CC
LL
YY
SS
KK
48
AA
LL
KK
HH
PP
VV
GG
RR
FF
WW
49
KK
MM
LL
CC
JJ
YY
HH
QQ
50
S
LL
CC
EE
JJ
AA
GG
QQ
MM
NN
KK
PP
TT
RR
FF
WW
HH
II
VV
XX
DD
51
SS
RR
TT
VV
AA
NN
HH
WW
EE
QQ
MM
GG
JJ
KK
DD
LL
52
EE
PP
53
LLAA
EE
GG
QQ
RR
YY
CC
TT
II
FF
JJ
NN
KK
MM
HH
PP
54
LL
PP
KK
RR
CC
NN
VV
JJ
XX
YY
II
55 56 57 58 59 60 61 62
SS
TT
LL
KK
RR
PP
NN
QQ
FF
MM
HH
GG
JJ
63 64
AAEE
65
SS
RR
KK
EE
NN
DD
JJ
AA
MM
TT
GG
LL
FF
CC
66
SS
VV
TT
JJ
RR
LL
NN
PP
WW
QQ
67
PP
WW
JJ
RR
VV
GG
LL
SS
QQ
68
SS
KK
RR
II
CC
LL
JJ
EE
FF
PP
VV
GG
AA
TT
HH
DD
XX
69
GGKK
RR
DD
WW
CC
TT
SS
LL
VV
MM
EE
FF
AA
YY
70
WW
CC
AA
LL
RR
QQ
VV
FF
KK
NN
II
71
KK
QQ
AA
CC
VV
TT
RR
GG
LL
HH
PP
SS
72
EE
GG
RR
JJ
PP
DD
HH
FF
MM
73
PP
JJ
WW
VV
AA
SS
74
VVRR
75
RR
VV
LLMM
EE
76
SS
KKTT
GG
CC 0
1
2
3
4
bits
1NN
QQ
23
NNTT
CC
4
RRMM
NN
WW
KK
YY
5
KK
VV
RR
LL
EEII
6
VV
LLRR
DD
GG
PP
AA
TT
7
KK
AA
VV
JJ
II
WW
89
10
SS
YY
11
SS
LL
QQDD
GG
WW
EE
12
JJ
SSVV
TT
RR
DD
GG
FF
AA
WW
II
MM
KK
NN
13
RR
QQ
GG
EE
14 15 16
PPKK
NN
MM
HH
QQ
AA
VV
RR
JJ
LL
SS
CC
YY
DD
TT
GG
FF
WW
17
RR
QQ
NN
VV
LL
TT
WW
DD
AA
FF
II
PP
18
RRJJPP
XX
19 20 21
DD
QQ
NN
LL
AA
VV
TT
FF
GG
XX
CC
WW
HH
KK
MM
YY
PP
II
SS
JJ
22
AA
VV
SS
JJ
RR
DD
II
HH
YY
23
SS
RR
EE
24
GG
LLJJ
25
RRSS
26
RR
GGSS
27
JJXX
28
GG
SS
DD
VV
29
RRLL
MM
GG
30
SS
RR
TT
QQ
JJ
HH
PP
KK
GG
WW
XX
31
DD
RR
TT
VV
GG
PP
LL
AA
KK
NN
32
LLKK
QQ
SS
RR
TT
GG
AA
PP
VV
EE
33
VV
SS
34
TT
LL
GG
PP
SS
CC
WW
35
SSRR
MM
LL
AA
TT
GG
VV
HH
QQ
PP
36 37
SS
RR
GG
TT
PP
VV
LL
AA
EE
QQ
KK
38
HH
YY
QQ
DD
KK
NN
EE
39
SS
RR
II
XX
MM
NN
TT
KK
JJ
40 41
VV
RR
LL
AA
PP
HH
QQ
42
RR
KK
SS
AA
NN
TT
LL
JJ
FF
HH
YY
43
PP
RRJJ
44
RR
TT
SS
QQ
PP
LL
VV
JJ
GG
45
AA
GG
PP
SS
WW
LL
EE
CC
HH
FF
II
46
TT
GG
JJ
CC
YY
LL
RR
SS
NN
KK
MM
47
RR
GG
HH
CC
MM
SS
LL
NN
KK
YY
48
KK
GG
VV
RR
JJ
FF
WW
49
RR
LL
KKJJ
QQ
HH
CC
YY
50
LL
KK
JJ
CCVV
RR
QQ
PP
GG
FF
DD
AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
QQ
JJ
GG
DD
LL
52 53
RR
KK
LL
QQ
NN
PP
TT
GG
SS
HH
YY
54
RR
NN
JJ
VV
QQ
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
RRTT
KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
QQ
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
QQ
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
QQ
FF
EE
70
RR
VV
QQ
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
function logos
Genome
g
tRNA
genes
T
g
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g tRNA CIFs are Nonetheless Highly
Evolutionarily Conserved tRNA Class Informative Features
(CIFs) are Based on Physics, not
Evolutionary Conservation
tRNA CIFs Predict Functional
Divergences Between Human and
Trypanosome aaRSs Rare tRNA CIFs are More
Functionally Informative scores Cyano-MLP
13
0.0
1.0
classification
probabilities0
1
2
3
4
bits
1
AA
SSMM
XX
JJ
FFNN
2
GG
JJ
CC
WW
YY
HH
3
GG
NN
VV
LL
CC
TT
EE
4
TT
JJ
EE
VV
AA
CC
RR
WW
HH
NN
MM
KK
YY
5
LL
GGKK
TT
JJ
FF
NN
AA
RR
YY
WW
II
6
VV
EE
MM
YY
JJ
KK
NN
HH
LL
RR
GG
AA
PP
DD
TT
7
SS
RR
GGNN
YY
KK
AA
VV
WW
JJ
II
89
GG
JJ
FF
10
SS
RRYY
11
RR
AA
NN
LL
TT
PP
YY
HHDD
EE
GG
WW
12
PP
JJSSWW
KK
VV
GG
MM
TT
NN
RR
AA
II
FF
DD
13
KK
YY
JJ
LL
CC
TT
AA
RR
MM
WW
GG
EE
14
SS
AA
YY
15
KK
QQJJ
16
PP
II
EE
KK
XX
HH
RR
SSNN
JJ
DD
WW
YY
VV
AA
TT
GG
FF
CC
MM
LL
17
SS
YY
HH
EE
NN
KK
TT
VV
RR
DD
FF
LL
WW
PP
AA
II
18
SS
TT
EEXX
PP
19 20 21
EE
JJ
YYLLDD
WW
KK
AA
TT
SS
MM
HH
XX
NN
FF
GG
PP
II
CC
VV
22
GG
PP
FFWW
NN
XX
CC
KK
EE
JJ
VV
SS
MM
HH
YY
DD
RR
II
23
PP
DD
NN
SS
RREE
24
YY
MM
JJ
LL
KK
25
KK
NN
FF
SS
QQYY
26
TTYY
KK
SS
27
MM
YYXX
28
SS
JJ
FF
EE
MM
VV
LL
AA
YY
TT
WW
KK
GG
RR
29
RR
LL
MM
NN
GG
WW
30
PP
VV
SS
LL
YY
JJ
FF
EE
WW
AA
MM
NNKK
TT
RR
CC
GG
XX
31
RRYY
WW
JJ
CC
EE
PP
TT
LL
DD
SS
GG
VV
FF
KK
NN
AA
HH
32
GGJJ
YY
DD
LL
MM
KK
SS
FF
TT
RR
AA
VV
PP
EE
33
AASS
34
VV
SS
JJ
EE
AA
MM
NN
TT
RR
DD
LL
GG
YY
XX
PP
FF
HH
CC
WW
35
KK
YYNN
FF
EE
MM
SS
RR
AA
GG
LL
VV
TT
HH
PP
36 37
SS
LL
TT
GG
PP
VV
RR
AA
KK
EE
38
NN
EE
HH
DD
YY
KK
39
SS
RR
KK
TT
XX
MM
II
JJ
NN
40AA
41
SSGG
RR
AA
PP
LL
VV
HH
42
NN
GG
EE
RR
AA
KK
HH
TT
YY
FF
LL
SS
JJ
43
TT
GG
LL
EE
DD
NN
AA
SS
MM
RR
YY
CC
JJ
44
FF
AA
NN
HH
JJ
KK
XX
MM
YY
RR
PP
VV
SS
LL
GG
45
KK
GG
PP
DD
MM
HH
RR
SS
EE
LL
YY
TT
FF
CC
WW
II
46
JJ
FF
MM
GG
YY
RR
WW
TT
CC
LL
SS
KK
NN
47
AAQQ
FF
DDTT
WW
RR
JJ
EE
GG
MM
HH
CC
LL
YY
KK
NN
SS
48
TT
KK
HH
SS
NN
AA
JJ
LL
CC
MM
PP
VV
GG
RR
FF
WW
49
RR
KK
FF
EE
SS
JJ
CC
HH
LL
YY
50
YY
JJ
SS
LL
EE
CC
GGQQMM
WW
AA
KK
RR
FF
TT
NN
VV
PP
XX
II
DD
HH
51
SS
PP
FF
CC
HH
JJ
VV
YY
TT
AA
NN
RR
MM
WW
EE
GG
KK
DD
LL
52
GG
II
KK
53
VV
JJ
AA
CC
MM
LL
KK
GG
RR
WW
YY
TT
SS
FF
PP
HH
NN
54
TT
MM
LL
PP
NN
JJ
VV
XX
YY
II
55
GG
NN
YY
56 57 58 59YY
60 61 62
AA
RRDD
EE
CC
XX
KK
LL
MM
YY
FF
NN
TT
GG
HH
JJ
63
DD
FF
TT
YY
AA
HH
NN
RR
MM
EE
JJ
LL
KK
VV
GG
II
XX
SS
CC
PP
WW
64
VVYYEE
65
RR
VV
SS
NN
AA
GG
YY
XX
DD
WW
FF
TT
LL
CC
66
AA
SS
YY
FF
MM
XX
TT
LL
JJ
VV
RR
KK
PP
WW
NN
67
GG
MM
KK
YY
WW
LL
SS
RR
68
SS
NNMM
FF
KK
LL
JJ
GG
PP
TT
EE
XX
VV
AA
HH
DD
69
RRSS
JJ
TT
MMLL
KK
DD
NN
WW
YY
VV
GG
XX
AA
EE
FF
CC
70
KK
SS
GG
WW
NN
LL
RR
JJ
DD
AA
MM
FF
VV
II
71
YY
FF
NN
KK
JJ
WW
AA
CC
TT
GG
RR
LL
VV
HH
PP
SS
72
SS
YY
TT
GG
AA
NN
XXKK
JJ
RR
PP
MM
HH
FF
DD
73
RRTT
NN
JJ
WW
YY
VV
AA
SS
74
YY
DD
NN
XX
WW
RR
75
TT
GGWW
NN
VV
RR
MM
LL
YY
XX
EE
76
SS
KK
JJ
MM
EE
YYGG
TT
CC 0
1
2
3
4
bits
1
RR
PP
SS
LL
KK
EE
FF
MM
JJ
XXNN
2
SS
PPJJ
II
MM
WW
CC
HH
3
SS
VV
EE
RR
FF
JJ
CC
II
MM
LL
TT
4
LL
SS
FF
WW
AA
II
VV
CC
RR
HH
JJNN
MM
KK
YY
5
PP
SS
DD
HH
GG
KK
RR
LL
NN
EE
AA
FFWW
II
6
SS
WW
CC
KK
NN
MM
HH
EE
JJ
VV
LL
RR
GG
PP
TT
AA
DD
7
LL
RR
TT
YY
SS
NN
MM
KK
VV
AA
WW
JJ
II
89
10
WWSSYY
11
VV
SS
LL
HH
PP
RR
QQEE
GG
DD
WW
12
EE
CC
PP
SS
JJ
WW
TT
MM
RR
KK
GG
VV
FF
AA
II
NN
DD
13
PP
AA
SSNN
DD
CC
KK
MM
JJ
YY
RR
WW
GG
EE
14 15
AA
SS
NN
YY
KK
MM
XX
JJ
16
II
PP
XX
EE
KK
JJ
WW
LL
MM
HH
RR
NN
YY
DD
VV
SS
TT
AA
GG
CC
FF
17
SS
TTNN
EE
KK
YY
HH
JJ
RR
MM
WW
VV
DD
FF
II
LL
PP
AA
18
TT
II
FF
JJ
YYMMPP
XX
19 20 21
EE
LL
JJ
NN
WW
MM
KK
AA
DD
SS
GG
TT
PP
CC
YY
VV
XX
FF
II
HH
22
LL
GG
KK
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36 37
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54
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63
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64
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65
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68
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69
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70
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71
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76
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CC 0
1
2
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13
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14 15 16
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18
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19 20 21
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24
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25
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26
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27
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28
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29
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30
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31
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32
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33
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35
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36 37
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38
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53
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54
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55
VV
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56 57 58 59 60 61 62
II
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63
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64
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65
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66
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67
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68
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69
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70
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71
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72
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73
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74
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75
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76
RRKKGG
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CC 0
1
2
3
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bits
1
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4
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5
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19 20 21
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31
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36 37
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39
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40 41
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47
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49
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50
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53
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54
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55
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56 57 58 59 60 61 62
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63
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64 65
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67
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68
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69
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70
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71
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72
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MM
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73
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74
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75
LLEE
76
TT
CC
GG 0
1
2
3
4
bits
1
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23
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4
RR
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MM
YY
5
LL
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14 15 16 17
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27
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28
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29 30
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31
AA
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32
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33 34
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35
NN
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36 37
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38
KK
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39
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40 41
RR
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42
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45
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46
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47
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48
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49
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50 51
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52 53
RR
JJ
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54
PP
RR
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55 56 57 58 59 60 61 62
LLTT
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GG
63 64EE
65
RR
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66
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67
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68
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69
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70
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71
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72
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73
RR
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AA
74RR
75
RR
LL
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76
QQTT
CC
GG 0
1
2
3
4
bits
1
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MM
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2
RRFF
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CC
3
GG
VV
LL
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EE
4
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5
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6
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89
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14 15 16
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17
SS
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18
VVPP
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19 20 21 22
TT
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MM
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23
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24
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25
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26
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27
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28
RR
GG
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29
VV
LL
30
LL
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31
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32
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33
VV
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34
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35
FF
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36 37
SS
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38
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39
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40 41
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42
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44
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45
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48
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50 51
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52
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53
SS
VV
RR
MM
WW
CC
NN
TT
PP
LL
GG
HH
KK
54
RR
NN
VV
XX
YY
II
55 56 57 58 59 60 61 62
LL
KK
RR
TT
HH
YY
NN
GG
FF
63 64
AAEE
65
SS
GG
NN
YY
LL
MM
TT
CC
FF
66
LLGG
FF
SS
VV
RR
MM
YY
TT
KK
PP
NN
JJ
WW
67
GG
VV
LL
SS
RR
XX
68
RR
LL
SS
JJ
MM
FF
GG
PP
VV
EE
TT
DD
HH
AA
69
SS
WW
VV
GG
EE
AA
CC
FF
70
TT
KK
GG
JJ
AA
RR
LL
DD
VV
MM
NN
II
71
FF
JJ
GG
MM
RR
WW
CC
AA
VV
TT
LL
HH
PP
SS
72
GG
TTJJ
MM
RR
PP
HH
FF
DD
73
RR
MM
VV
JJ
SS
AA
74
WW
RR
75
RR
WW
LL
YYEE
76
VV
EE
WW
QQGG
TT
CC 0
1
2
3
4
bits
1
JJ
DDQQ
NN
2HH
3
GG
VV
EE
LL
MM
DD
JJ
TT
4
AA
FF
RR
DD
CC
HH
WW
NN
KK
YY
5
AA
KK
LL
FF
HH
EE
TT
NN
WW
RR
DD
II
6
VV
KK
MM
NN
LL
EE
HH
CC
DD
TT
AA
RR
GG
PP
7
SS
KK
EE
AA
MM
JJ
VV
WW
II
89
10 11
RR
PP
LL
HH
GG
DD
EE
WW
12
PP
SS
KK
JJ
TT
AA
GG
WW
VV
RR
II
MM
NN
FF
DD
13
LL
SS
AAWW
DD
EE
GG
14 15 16
PP
II
EE
MM
KK
TT
XX
NN
DDHH
YY
CC
VV
AA
FF
JJ
LL
GG
RR
SS
WW
17
SS
KK
EE
TT
QQMM
RR
LL
DD
VV
PP
FF
WW
AA
II
18
RRJJ
PP
XX
19 20
DD
RR
21 22
GG
KK
EE
CC
VV
SS
MM
RR
HH
DD
II
YY
23
SSRR
DD
CCEE
24
SS
LL
25
SS
FF
NNYY
26
KKTT
SS
27
EE
JJ
XX
28
LLGG
FF
AA
VV
KK
29
LL
GG
30
LL
EE
PP
WW
SS
RR
AA
TT
JJ
VV
KK
GG
XX
31
PP
RR
EE
TT
JJ
FF
YY
SS
AA
LL
VV
HH
KK
NN
32
SS
YY
CC
LL
RR
FF
VV
AA
TT
PP
GG
EE
33
SS
LL
MM
34
GG
RR
YY
LL
SS
TT
EE
PP
FF
HH
CC
MM
WW
35
SS
RR
EE
AA
LL
GG
HH
VV
TT
PP
36 37
SS
RR
TT
GG
LL
VV
AA
KK
PP
EE
38
KK
YY
NN
EE
HH
DD
39
SS
RR
XX
MM
KK
NN
JJ
TT
II
40 41
RR
AA
PP
VV
LL
HH
42
GG
RR
EE
HH
AA
TT
KK
NN
JJ
LL
FF
SS
YY
MM
43
AA
LL
HH
44
MM
JJ
AA
WW
RR
PP
SS
GG
CC
VV
LL
45
PP
EE
GG
LL
MM
YY
SS
JJ
FF
HH
WW
CC
II
46
EE
GG
VV
LL
TT
SS
RR
NN
JJ
KK
CC
MM
47
VV
MM
HH
WW
GG
NN
CC
LL
YY
SS
KK
48
AA
LL
KK
HH
PP
VV
GG
RR
FF
WW
49
KK
MM
LL
CC
JJ
YY
HH
50
S
LL
CC
EE
JJ
AA
GG
MM
NN
KK
PP
TT
RR
FF
WW
HH
II
VV
XX
DD
51
SS
RR
TT
VV
AA
NN
HH
WW
EE
MM
GG
JJ
KK
DD
LL
52
EE
PP
53
LLAA
EE
GG
RR
YY
CC
TT
II
FF
JJ
NN
KK
MM
HH
PP
54
LL
PP
KK
RR
CC
NN
VV
JJ
XX
YY
II
55 56 57 58 59 60 61 62
SS
TT
LL
KK
RR
PP
NN
FF
MM
HH
GG
JJ
63 64
AAEE
65
SS
RR
KK
EE
NN
DD
JJ
AA
MM
TT
GG
LL
FF
CC
66
SS
VV
TT
JJ
RR
LL
NN
PP
WW
67
PP
WW
JJ
RR
VV
GG
LL
SS
68
SS
KK
RR
II
CC
LL
JJ
EE
FF
PP
VV
GG
AA
TT
HH
DD
XX
69
GGKK
RR
DD
WW
CC
TT
SS
LL
VV
MM
EE
FF
AA
YY
70
WW
CC
AA
LL
RR
VV
FF
KK
NN
II
71
KK
AA
CC
VV
TT
RR
GG
LL
HH
PP
SS
72
EE
GG
RR
JJ
PP
DD
HH
FF
MM
73
PP
JJ
WW
VV
AA
SS
74
VVRR
75
RR
VV
LLMM
EE
76
SS
KKTT
GG
CC 0
1
2
3
4
bits
1NN
23
NNTT
CC
4
RRMM
NN
WW
KK
YY
5
KK
VV
RR
LL
EEII
6
VV
LLRR
DD
GG
PP
AA
TT
7
KK
AA
VV
JJ
II
WW
89
10
SS
YY
11
SS
LL
QQDD
GG
WW
EE
12
JJ
SSVV
TT
RR
DD
GG
FF
AA
WW
II
MM
KK
NN
13
RR
GG
EE
14 15 16
PPKK
NN
MM
HH
AA
VV
RR
JJ
LL
SS
CC
YY
DD
TT
GG
FF
WW
17
RR
NN
VV
LL
TT
WW
DD
AA
FF
II
PP
18
RRJJPP
XX
19 20 21
DD
NN
LL
AA
VV
TT
FF
GG
XX
CC
WW
HH
KK
MM
YY
PP
II
SS
JJ
22
AA
VV
SS
JJ
RR
DD
II
HH
YY
23
SS
RR
EE
24
GG
LLJJ
25
RRSS
26
RR
GGSS
27
JJXX
28
GG
SS
DD
VV
29
RRLL
MM
GG
30
SS
RR
TT
JJ
HH
PP
KK
GG
WW
XX
31
DD
RR
TT
VV
GG
PP
LL
AA
KK
NN
32
LLKK
SS
RR
TT
GG
AA
PP
VV
EE
33
VV
SS
34
TT
LL
GG
PP
SS
CC
WW
35
SSRR
MM
LL
AA
TT
GG
VV
HH
PP
36 37
SS
RR
GG
TT
PP
VV
LL
AA
EE
KK
38
HH
YY
DD
KK
NN
EE
39
SS
RR
II
XX
MM
NN
TT
KK
JJ
40 41
VV
RR
LL
AA
PP
HH
42
RR
KK
SS
AA
NN
TT
LL
JJ
FF
HH
YY
43
PP
RRJJ
44
RR
TT
SS
PP
LL
VV
JJ
GG
45
AA
GG
PP
SS
WW
LL
EE
CC
HH
FF
II
46
TT
GG
JJ
CC
YY
LL
RR
SS
NN
KK
MM
47
RR
GG
HH
CC
MM
SS
LL
NN
KK
YY
48
KK
GG
VV
RR
JJ
FF
WW
49
RR
LL
KKJJ
HH
CC
YY
50
LL
KK
JJ
CCVV
RR
PP
GG
FF
DD
AA
XX
WW
MM
II
HH
NN
TT
51
TT
RR
JJ
GG
DD
LL
52 53
RR
KK
LL
NN
PP
TT
GG
SS
HH
YY
54
RR
NN
JJ
VV
II
XX
YY
55
RRMM
56 57 58 59 60 61 62
RRTT
KK
VV
HH
GG
JJ
63 64
AA
65
SS
RR
VV
LL
GG
NN
TT
FF
CC
66
LL
TT
VV
JJ
RR
PP
NN
WW
67
PP
VV
WW
GG
SS
DD
JJ
LL
RR
KK
68
LL
RR
MM
VV
JJ
GG
TT
FF
PP
DD
EE
HH
XX
AA
69
GG
TTVV
DD
SS
CC
AA
YY
FF
EE
70
RR
VV
LLII
MM
71
GG
VV
AA
RR
LL
TT
PP
SS
HH
72
SS
RR
LL
DD
PP
MM
FF
73
RRVVAA
SS
74
DD
75
JJXX
EE
76
CC
TT
GG
function logos
Genome
g
tRNA
genes
T
g
D
A
S
g
B1
S
g
B2+3
S
g
C1
S
g
C3
Sg
E
S
g
F
S
g
G
S
g tRNA CIFs are Nonetheless Highly
Evolutionarily Conserved tRNA Class Informative Features
(CIFs) are Based on Physics, not
Evolutionary Conservation
CIF Divergence Models Predict Gains and Conversions of
tRNA CIFs in Leishmania parasites, relative to humans.
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
tRNA CIFs in Leishmania parasites, relative to humans.
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
Based on our models, Paul Kelly from Mike Ibba’s lab was able to find
parasite-specific AlaRS inhibitors from a Marine Natural Products Library
A
C D
B
Identification of Leishmania
major AlaRS inhibitors.
A) Workflow to identify
aminoacylation inhibitors
B) Representative image of the
MNP chemical screen. The spot
boxed in red is an example of a
predicted inhibitor depicted by the
decrease in signal intensity. DMSO
positive control (+).
C) Three of the four identified
inhibitors prevented the
accumulation of Ala-tRNA
Ala
formation.
D) The three identified
inhibitors perturbed L. major
AlaRS activation (black) but had
no effect on human AlaRS (gray).
The relative amino acid activation is
plotted relative to the DMSO
control. Error bars indicate the
standard deviation of three
replicates.
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
parasite-specific AlaRS inhibitors from a Marine Natural Products Library
A
C D
B
Identification of Leishmania
major AlaRS inhibitors.
A) Workflow to identify
aminoacylation inhibitors
B) Representative image of the
MNP chemical screen. The spot
boxed in red is an example of a
predicted inhibitor depicted by the
decrease in signal intensity. DMSO
positive control (+).
C) Three of the four identified
inhibitors prevented the
accumulation of Ala-tRNA
Ala
formation.
D) The three identified
inhibitors perturbed L. major
AlaRS activation (black) but had
no effect on human AlaRS (gray).
The relative amino acid activation is
plotted relative to the DMSO
control. Error bars indicate the
standard deviation of three
replicates.
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
tRNA
Ala CIF Divergences between trypanosomes and humans are
conserved over 250 my of trypanosome evolution.
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
Ala CIF Divergences between trypanosomes and humans are
conserved over 250 my of trypanosome evolution.
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
Significance of CIF Divergences between Trypanosomes and Humans
Lawrence et al. (2021). Bioinformatics 37(20):3654
Lawrence et al. (2021). Bioinformatics 37(20):3654
Correspondingly, Paul found that the inhibitors we found against
Leishmania AlaRS also worked against Trypanosoma AlaRS
Leishmania major and
Trypanosoma cruzi AlaRS have
conserved tRNA identity
elements.
A) CIF Divergence Models for
tRNAAla in Leishmania major and
Trypanosoma cruzi
B) The three identified Lm AlaRS
inhibitors also have activity against
the Tc AlaRS enzyme. Error bars
indicate the standard deviation of
three replicates.
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
Leishmania AlaRS also worked against Trypanosoma AlaRS
Leishmania major and
Trypanosoma cruzi AlaRS have
conserved tRNA identity
elements.
A) CIF Divergence Models for
tRNAAla in Leishmania major and
Trypanosoma cruzi
B) The three identified Lm AlaRS
inhibitors also have activity against
the Tc AlaRS enzyme. Error bars
indicate the standard deviation of
three replicates.
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
Paul Kelly also found multiple parasite-specific
ThrRS inhibitors from the same Marine Natural Products Library
A
B C
Identification of Leishmania
major ThrRS inhibitors.
A) The MNP library was re-
screened at the highest
concentrations to qualitatively
identify Lm
ThrRS aminoacylation
inhibitors. Plate IDs reference
the position within the original
library and not library IDs.
B) Eight inhibitors were
qualitatively identified from the
preliminary screen. Two of the
candidates did not inhibit
aminoacylation (black), two
inhibited at ~50% activity
(gray), and four inhibited at
greater than 25% (white).
C) All four active inhibitors
continued to perturb
aminoacylation over a time
course experiment. Error
bars indicate the standard
deviation of three replicates.
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
ThrRS inhibitors from the same Marine Natural Products Library
A
B C
Identification of Leishmania
major ThrRS inhibitors.
A) The MNP library was re-
screened at the highest
concentrations to qualitatively
identify Lm
ThrRS aminoacylation
inhibitors. Plate IDs reference
the position within the original
library and not library IDs.
B) Eight inhibitors were
qualitatively identified from the
preliminary screen. Two of the
candidates did not inhibit
aminoacylation (black), two
inhibited at ~50% activity
(gray), and four inhibited at
greater than 25% (white).
C) All four active inhibitors
continued to perturb
aminoacylation over a time
course experiment. Error
bars indicate the standard
deviation of three replicates.
Kelly et al. (2020) PLoS Negl. Trop. Dis. 14(2): e0007983.
Fatemeh Hadi-
Nezhad
Travis Lawrence, Ph.D.
Machine Learning
Division
FlyingJ
Prof. Dr. Ivo Grosse
Martin-Luther-Universität, Halle-
Wittenberg
NSF MRI OAC-1429783
Nezhad
Travis Lawrence, Ph.D.
Machine Learning
Division
FlyingJ
Prof. Dr. Ivo Grosse
Martin-Luther-Universität, Halle-
Wittenberg
NSF MRI OAC-1429783
Mike Ibba Lab
(OSU/ Chapman U.)
Paul Kelly
Roger Linington Lab (SFU)
Dennis Liu
AI127582
NSF MRI OAC-1429783
Fatemeh Hadi-
Nezhad
Travis Lawrence, Ph.D.
Machine Learning
Division
FlyingJ
(OSU/ Chapman U.)
Paul Kelly
Roger Linington Lab (SFU)
Dennis Liu
AI127582
NSF MRI OAC-1429783
Fatemeh Hadi-
Nezhad
Travis Lawrence, Ph.D.
Machine Learning
Division
FlyingJ
Julie Phillips, Ph.D.
Professor
Cumberland University
NSF MRI OAC-1429783
Professor
Cumberland University
NSF MRI OAC-1429783
Wes Swingley, Ph.D
Professor NIU
Katie Amrine, Ph.D.
Decision Science Manager
Meta
NSF INSPIRE BCS-1344279
NSF MRI OAC-1429783
Travis Lawrence, Ph.D.
Machine Learning
Division
FlyingJ
Professor NIU
Katie Amrine, Ph.D.
Decision Science Manager
Meta
NSF INSPIRE BCS-1344279
NSF MRI OAC-1429783
Travis Lawrence, Ph.D.
Machine Learning
Division
FlyingJ
Other tRNA classes, but not all,
show evidence of Conserved Divergence in CIFs
Major (n = 8) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Infantum (n = 3) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Mexicana (n = 2) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Viannia (n = 4) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Af. Tryp. (n = 6) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Am. Tryp. (n = 11) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Lepto/Crith (n = 3) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Enriettii (n = 2) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Major (n = 8) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Infantum (n = 3) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Mexicana (n = 2) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Viannia (n = 4) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Af. Tryp. (n = 6) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Am. Tryp. (n = 11) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Lepto/Crith (n = 3) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Enriettii (n = 2) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Major (n = 8) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Infantum (n = 3) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Mexicana (n = 2) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Viannia (n = 4) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Af. Tryp. (n = 6) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Am. Tryp. (n = 11) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Lepto/Crith (n = 3) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Enriettii (n = 2) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Major (n = 8) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Infantum (n = 3) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Mexicana (n = 2) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Viannia (n = 4) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Af. Tryp. (n = 6) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Am. Tryp. (n = 11) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Lepto/Crith (n = 3) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Enriettii (n = 2) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
show evidence of Conserved Divergence in CIFs
Major (n = 8) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Infantum (n = 3) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Mexicana (n = 2) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Viannia (n = 4) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Af. Tryp. (n = 6) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Am. Tryp. (n = 11) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Lepto/Crith (n = 3) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Enriettii (n = 2) K
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Major (n = 8) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Infantum (n = 3) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Mexicana (n = 2) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Viannia (n = 4) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Af. Tryp. (n = 6) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Am. Tryp. (n = 11) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Lepto/Crith (n = 3) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Enriettii (n = 2) S
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Major (n = 8) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Infantum (n = 3) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Mexicana (n = 2) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Viannia (n = 4) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Af. Tryp. (n = 6) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Am. Tryp. (n = 11) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Lepto/Crith (n = 3) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Enriettii (n = 2) V
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Major (n = 8) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Infantum (n = 3) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Mexicana (n = 2) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Viannia (n = 4) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Af. Tryp. (n = 6) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Am. Tryp. (n = 11) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Lepto/Crith (n = 3) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
Enriettii (n = 2) Y
55
60
65
70
15
18
35
45
50
10
14
25
21
3040
5'
3'
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