Next Generation Sequence Analysis and genomics

Next Generation Sequence Analysis and genomics

1. Next Generation
Sequencing
By: SajadRafatiyan
Faculty of Advanced Sciences and Technologies
University of Isfahan

2. Contents
•Introduction
•NGS Tools
•NGS Machines
•Different methods of NGS
•Sequencing by Ligation
•Illumina sequencing
•454 sequencing
•Ion Torrent: Proton / PGM sequencing
•Single Molecule Real-Time Sequencing (SMRT)
•GradIONNanopore Sequancing
•Sources

3. Introduction
In 1953 James Watson and Francis Crick
described the double helix of DNA.
In 1977 Frederick Sanger invented a
method for sequencing the DNA
sequence using dideoxynucleosides.
Through this method and via using
fluorescence tagged nucleotides and
chromatography, sequencing 600-800
bpsequences is possible.

4. In 1960 Thomas Dale Brock reported
hyperthermophiles living in hot springs at
Yellowstone National Park.
After that in 1976 Chienet al isolated
thermostable DNA polymerase form
Thermusaquaticus, and now we use it
for PCR.
PCR (Polymerase Chain Reaction) was
Develop in 1983 by KaryMullis.

5. In 1990 Illumina/solax, Roch/454 Life Science and ABI/SoliDmade some machines for
DNA sequencing separately.
The machines have been commercialyavailable in 2005-2006.
Bioinformatic tools can be used for sequence analysis and the whole genome of
organism is sequenced.

6. NGS Tools
Visualization
Integrative Genomics Viewer (IGV)
Artemis
Abrowse
Integrated Genome Browser (IGB)

7. At first it was really expansive to sequencing a DNA molecule, efficiency
was not really good and it took long time to sequencing a molecule.
Today Illumina has been insisted which can
sequencing human genome in one hour by
100 dollars

8. NGS Machines

11. Different methods of sequencing
1.Pyrosequencing
2.Sequencing by Synthesis
3.Sequencing by Ligation
4.Ion Semiconductor Sequencing

12. Sequencing by Ligation

13. Sequencing by Ligation

15. Illumina sequencing
In NGS, vast numbers of short reads are sequenced in a single stroke.
To do this, firstly the input sample must be cleaved into short sections.
The length of these sections will depend on the particular sequencing
machinery used.
In Illumina sequencing, 100-150bpreads are used. Somewhat longer
fragments are ligated to generic adaptors and annealed to a slide using
the adaptors. PCR is carried out to amplify each read, creating a spot with
many copies of the same read. They are then separated into single
strands to be sequenced.

16. The slide is flooded with nucleotides and
DNA polymerase. These nucleotides are
fluorescently labelled, with the color
corresponding to the base. They also
have a terminator, so that only one base
is added at a time.

17. An image is taken of the slide. In
each read location, therewill be
a fluorescent signal indicating
the base that has been added.

18. The slide is then prepared for the next cycle. The terminators are
removed, allowing the next base to be added, and the fluorescent
signal is removed, preventing the signal from contaminating the next
image.
The process is repeated, adding one nucleotide at a time and imaging
in between.

19. Computers are then used to detect the base at each site in each image
and these are used to construct a sequence.
All of the sequence reads will be the same length, as the read length
depends on the number of cycles carried out.

20. 454 sequencing
Roche 454 sequencing can sequence much longer reads than Illumina. Like
Illumina, it does this by sequencing multiple reads at once by reading
optical signals as bases are added.
As in Illumina, the DNA or RNA is fragmented into shorter reads, in this case
up to 1kb. Generic adaptors are added to the ends and these are annealed
to beads, one DNA fragment per bead. The fragments are then amplified by
PCR using adaptor-specifcprimers.
Each bead is then placed in a single well of a slide. So each well will contain
a single bead, covered in many PCR copies of a single sequence. The wells
also contain DNA polymerase and sequencing buffers.

21. The slide is flooded with one of the
four NTP species. Where this
nucleotide is next in the sequence, it
is added to the sequence read. If that
single base repeats, then more will
be added. So if we flood with
Guanine bases, and the next in a
sequence is G, one G will be added,
however if the next part of the
sequence is GGGG, then four Gswill
be added.

22. The addition of each nucleotide releases a
light signal. These locations of signals are
detected and used to determine which
beads the nucleotides are added to.

23. This NTP mix is washed away. The next NTP mix is now added and the
process repeated, cycling through the four NTPs.

24. This kind of sequencing generates
graphs for each sequence read,
showing the signal density for each
nucleotide wash. The sequence can
then be determined computationally
from the signal density in each wash.
All of the sequence reads we get from
454 will be different lengths, because
different numbers of bases will be
added with each cycle.

25. Ion Torrent: Proton / PGM sequencing
Unlike Illumina and 454, Ion torrent and Ion proton sequencing do not make use of
optical signals. Instead, they exploit the fact that addition of a dNTP to a DNA polymer
releases an H+ ion.
As in other kinds of NGS, the input DNA or RNA is fragmented, this time ~200bp.
Adaptors are added and one molecule is placed onto a bead. The molecules are
amplified on the bead by emulsion PCR. Each bead is placed into a single well of a
slide.

26. Like 454, the slide is flooded with a single
species of dNTP, along with buffers and
polymerase, one NTP at a time. The pH is
detected is each of the wells, as each H+
ion released will decrease the pH.The
changes in pH allow us to determine if
that base, and how many thereof, was
added to the sequence read.

27. The dNTPs are washed away, and the process is repeated cycling
through the different dNTP species.

28. The pH change, if any, is used to determine how many bases (if any)
were added with each cycle.

31. SMRT (Single Molecule Real-Time sequencing)

32. SMRT Sequencing

33. Advantages SMRT Sequencing
•Deconvolute complex mixtures of unique haplotypes
•Accurately identify somatic variants
•Resolve complex communities

35. GridIONNanopore

37. Application of NGS
•Whole Genome Sequencing (to find point mutationor be sure about gene integrationin right
place)
•Target Sequencing (hotspot sequences mutation for canceror immune system disease)
•De Novo Sequencing and Assembly (for new organismwhich have not enough information
about them)
•RNA-Sequencing (to detect codingand non-codingsequences and sometime we can use it as
genome sequence)
•Epigenetic changes

38. Application of NGS
•Single cell sequencing
•Free DNA sequencing (detection of canceror genetic disordersbefore birth)
•Long non-coding RNA interactions (fore gene translation regulation)
•For Methylation Assisted Isolation of Regulatory Elements (FAIREDNasesequencing)

39. Sources
Wong, L.-J. C., 2013. Next Generation Sequencing: Translation to Clinical Diagnostics.
1st ed. NewYork: springer
Jay Shendure& HanleeJi, 2008. Next-generation DNA sequencing. Nature
Biotechnology, 26(10).
https://www.genewiz.com/en/Public/Services/Next-Generation-Sequencing
https://www.ebi.ac.uk/training/online/course/ebi-next-generation-sequencing-
practical-course
https://nanoporetech.com/applications/dna-nanopore-sequencing

40. Thank you for your attention