Next Generation Sequencing and its application

Next Generation Sequencing and its application

1. 1
Next Generation Sequencing

2. 2
2010: 5K$,
a few days?
2009: Illumina,
Helicos
40-50K$
Sequencing the Human Genome
Year
Log
10
(price)
201020052000
10
8
6
4
2
2012: 100$, <24
hrs?
2008: ABI SOLiD
60K$, 2 weeks
2007: 454
1M$, 3 months
2001: Celera
100M$, 3 years
2001: Human Genome Project
2.7G$, 11 years

3. 3
Content:
•Sequencing 1.0: Sanger
•Assembly
•Next generation sequencing (NGS)
•NGS applications
•Future directions

4. Genome Sequencing
•Goal
figuring the order of nucleotides across a genome
•Problem
Conventional DNA sequencing methods can
handle only short stretches of DNA at once (<1-
2Kbp)
•Solution
Sequence and then use computers to assemble
the small pieces
4

5. Genome Sequencing
5
5
ACGTGGTAA CGTATACAC TAGGCCATA
GTAATGGCG CACCCTTAG
TGGCGTATA CATA…
ACGTGGTAATGGCGTATACACCCTTAGGCCATA
Short fragments of DNA
AC..GC
TT..TC
CG..CA
AC..GC
TG..GT TC..CC
GA..GC
TG..AC
CT..TG
GT..GC AC..GC AC..GC
AT..AT
TT..CC
AA..GC
Short DNA sequences
ACGTGACCGGTACTGGTAACGTACA
CCTACGTGACCGGTACTGGTAACGT
ACGCCTACGTGACCGGTACTGGTAA
CGTATACACGTGACCGGTACTGGTA
ACGTACACCTACGTGACCGGTACTG
GTAACGTACGCCTACGTGACCGGTA
CTGGTAACGTATACCTCT...
Sequenced genome
Genome

6. DNA Sequencing
•Theprocessofdeterminingtheorderofbases
adenine(A),thymine(T),cytosine(C),andguanine
(G)alongaDNAstrand.
•Alltheinformationrequiredforthegrowthand
developmentofanorganismisencodedintheDNA
ofitsgenome.
•So,DNAsequencingisfundamentaltogenome
analysisandunderstandingthebiologicalprocesses
ingeneral.

7. Technical Breakthrough For
DNA Sequencing
In 1977, two separate methods for the
large-scale sequencing of DNA were
devised:
•Chemical cleavage method
by A. M. Maxam and W. Gilbert
•Enzymatic chain termination method
by F. Sanger et. al.

8. A.Ofthesetwomethods,Sangermethodismore
popular.Withoutchangingtheunderlyingconcept
ofbothmethods,someimprovementshavebeen
doneovertheyearsbyapplyingdifferentstrategies,
bydevelopingvariousmodificationsandby
automation.
B.Asaresult,averylargescalesequencinghas
becomefeasible,e.g.E.coli,Saccharomyces
cerevisiae,HumanGenomeProjectetc.

9. Chemical Cleavage Method
•This method uses double-stranded DNA samples.
•Involves modification of the bases in DNA followed by
chemical base-specific cleavage.
•Sequences DNA fragments containing upto ~500
nucleotides in length.

10. Stages:
1.The double-stranded fragment to be
sequenced is isolated and radioactively
labeled at the 5’-ends with
32
P.
2.The fragment is then cut with restriction
enzyme and thus the label is removed
from one end.
3.The fragment of DNA with one end
labeled is denatured.
4.Four identical samples of these end-
labeled DNA restriction fragments are
subjected to chemical cleavage at
different chemical nucleotides.
5.There are four specific sets of chemical
reactions that selectively cut the DNA
backbone at G, A+G, C+T, or C residues.
–G only: Dimethyl sulphate(DMS)
and piperidine
–A+G : DMS, piperidine
–C+T : Hydrazine, piperidine
–C only : Hydrazine, alkali,
piperidine
Figure: Maxam-Gilbert method
(continued)
Lodish, H.;Berk, A. et. al. (4
th
ed);
Mol. Cell Biol.; W. H. Freeman and Co. (2000)
p: 233

11. 6.For each labeled chain to be
broken only once, the reactions
are controlled.
7.The labeled subfragments
created by the four reactions
have
–the
32
P label at one end and
–the chemical cleavage point
at the other end.
8.The reaction products are
separated by polyacrylamide gel
electrophoresis which is based
on size. Smallest fragment goes
fastest.
Figure: Apparatus for gel electrophoresis
Voet, D.; Voet, J. and Pratt, C. (upgrade ed)
Fundamentals of Biochemistry; John Wiley and Sons, Inc
(2002); p: 58

12. 9. The labeled fragments
in the gel are visualized
by autoradiography.
10. The sequence is read
from bottom to top of
the gel.
Figure: Maxam-Gilbert method
Lodish, H.;Berk, A. et. al. (4th ed);
Mol. Cell Biol.; W. H. Freeman and Co. (2000)
p: 233

13. Example of DNA Sequencing by
Chemical Method
http://users.wmin.ac.uk/~redwayk/lectures/sequence.htm

14. Mechanism of the chemical cleavage
method
Voet, D.; Voet, J. Biochemistry; John Wiley and Sons, Inc (1990); p: 830

15. Continued
Voet, D.; Voet, J. Biochemistry; John Wiley and Sons, Inc (1990); p: 831

16. Advantages Disadvantages
•No premature termination due
to DNA sequencing. So, no
problem with polymerase to
synthesize DNA.
•Stretches of DNA can be
sequenced which can not be
done with enzymatic method.
•Not widely used.
•Use of radioactivity and
toxic chemicals.
http://www.cmb.uab.edu/courses/lectures/scheirer2.pdf

17. Chain Termination method
•This method uses single-stranded DNA.
•Also known as dideoxy sequencingmethod because it involves the
use of analogue of normal nucleotide 2’,3’-dideoxynucleoside
triphosphates (ddNTPs). These are chain terminating nucleotides
lacking 3’-OH ends.
•This method is based upon the incorporation of ddNTPs into a
growing DNA strand to stop chain elongation.
Figure: Structure of NTP,
dNTP, and ddNTP
Lodish, H.;Berk, A. et. al. (4th ed);
Mol. Cell Biol.; W. H. Freeman and Co. (2000), p:
233

18. Stages:
1.The DNA to be sequenced is called the template DNA. It is prepared as a single-stranded DNA
after being spliced into M13 vector DNA. Infected E. colihost cells release phage particles
which contains single-stranded recombinant DNA that includes the sample DNA. This DNA
sample is then extracted from phage for sequencing purpose.
2. A synthetic 5’-end-labeled oligodeoxynucleotide is used as the primer.
3. The template DNA is hybridized to the primer.
4. The primer elongation is performed in four separate polymerization reaction mixtures. Each
mixture contains
-4 normal deoxynucleotides (dNTPs)
in higher concentration and
-a low concentration of the each of
the 4 ddNTPs.
5. There is initiation of
DNA synthesis by adding
enzyme DNA polymerase
since the enzyme cannot
distinguish between the
normal nucleotides and
their analogues.
Figure: Action of DNA polymerase I
Voet, D.; Voet, J. and Pratt, C. (upgrade ed)
Fundamentals of Biochemistry; John Wiley and Sons, Inc (2002); p: 60

19. 6. The strand synthesis continues
until a ddNTP is added. The
chain elongation ceases on the
incorporation of a ddNTP
because it lacks a 3’-OH group
which prevents addition of the
next nucleotide.
7. There is a result of mixture of
terminated fragments, all of
different lengths.
8. Denature DNA fragments.
9. Each of the four mixtures are run
together on a polyacrylamide gel
for electrphoresis.
Figure: Sanger method
Lodish, H.;Berk, A. et. al. (4th ed);
Mol. Cell Biol.; W. H. Freeman and Co. (2000)
p: 234

20. 10. The separated
fragments are
then visualized
by autography.
11. From the position
of the bands of the resulting
autoradiogram, the
sequence of the original
DNA template strand can
be read directly.
Figure: Chain termination method
Voet, D.; Voet, J. and Pratt, C. (upgrade ed)
Fundamentals of Biochemistry; John Wiley and Sons, Inc (2002);
p: 61

21. •Most popular method.
•Simpler and quicker allowing
large output. Within an hour
the primer-annealing and
sequencing reactions can be
completed.
•Yielding of poor results owing
to secondary structure in the
DNA as sometimes DNA
polymerases terminate chain
elongation prematurely.
•The sequence is obtained not
from the original DNA molecule
but from an enzymatic copy. So,
there is a chance of
incorporation of wrong bases.
http://www.ich.ucl.ac.uk/cmgs/sequence.htm
Advantage
Disadvantage

22. Example of DNA Sequencing in
Sanger Method
http://users.wmin.ac.uk/~redwayk/lectures/sequence.htm

23. Other Improved Approaches and Automated DNA Sequencing
•Updated version of Sanger method
•Fluorescence detection with lasers
•Cycle sequencing
•Shotgun sequencing
http://www.cmb.uab.edu/courses/lectures/scheirer2.pdf
chsfpc5.chem.ncsu.edu/Poznan/ chem_bio/sld026.htm

24. opbs.okstate.edu/.../ sld015.htm

25. 
many pieces
to assemble
High coverage:
Assembly: How Much DNA?
25
Low coverage:
A few pieces
to assemble
a few contigs,
a few gaps
many contigs,
many gaps


Input Output
Lander and Waterman,
1988

26. Sanger Sequencing
26
1980 1990 2000
1982: lambda virus
DNA stretches up to
30-40Kbp
(Sanger et al.)
1994: H. Influenzae
1.8 Mbp
(Fleischmann et al.)
2001: H. Sapiens,
D. Melanogaster
3 Gbp
(Venter et al.)
2007: Global Ocean
Sampling Expedition
~3,000 organisms,
7Gbp (Venter et al.)

27. 27
Next Generation Sequencing:
Why Now?
•Motivation:HGP and its derivatives,
personalized medicine
•Short reads applications:(re-)sequencing,
other methods (e.g. gene expression)
•Advancements in technology

28. 28
High Parallelism is Achieved in
Polony Sequencing
PolonySanger

29. 29
Generation of Polony array:
DNA Beads (454, SOLiD)
DNA Beads are generated using Emulsion PCR

30. 30
Generation of Polony array:
DNA Beads (454, SOLiD)
DNA Beads are placed in wells

31. 31
Generation of Polony array:
Bridge-PCR (Solexa)
DNA fragments are attached to array and
used as PCR templates

32. 32
Sequencing: Pyrosequencing
(454)
Complementary strand elongation: DNA Polymerase

33. 33
Sequencing: Fluorescently
labeled Nucleotides (Solexa)
Complementary strand elongation: DNA Polymerase

34. 34
Sequencing: Fluorescently
Labeled Nucleotides (ABI SOLiD)
Complementary strand elongation: DNA Ligase

35. 35
Sequencing: Fluorescently
Labeled Nucleotides (ABI SOLiD)
5 reading frames, each position is read twice

36. 36
Single Molecule Sequencing:
HeliScope
•Direct sequencing of DNAmolecules: no
amplification stage
•DNA fragments are attached to array
•Potential benefits: higher throughput, less
errors

37. 37
Technology Summary
Read lengthSequencing
Technology
Throughput
(per run)
Cost (1mbp)*
Sanger ~800bp Sanger 400kbp 500$
454 ~400bp Polony 500Mbp 60$
Solexa 75bp Polony 20Gbp 2$
SOLiD 75bp Polony 60Gbp 2$
Helicos 30-35bp Single
molecule
25Gbp 1$
*Source: Shendure& Ji, Nat Biotech, 2008

38. 38
What, When and Why
•Sanger:
Small projects (less than 1Mbp)
•454:
De-novo sequencing, metagenomics
•Solexa, SOLiD, Heliscope:
–Gene expression, protein-DNA interactions
–Resequencing

39. 39
Applications

40. 40
Applications

41. 41
Where Do We Go from Here?
•Higher throughput, longer reads (Pacific
BioSciences)
•Computational bottleneck
•Shift to sequencing-based technologies
•Will it help to cure cancer?