In this lecture tried to introduce some basic methods of DNA sequencing like pyrosequencing, sequencing by ligation, sequencing by synthesis and Ion Semiconductor Sequencing and describe them. Also introduced some new sequencing method (third generation sequencing) like SMRT (Single Molecule Real-Time Sequencing) and GridION.

1. Next Generation
Sequencing
By: Sajad Rafatiyan
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)
•GradION Nanopore 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
bp sequences is possible.

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

5. In 1990 Illumina/solax, Roch/454 Life Science and ABI/SoliD made some machines for
DNA sequencing separately.
The machines have been commercialy available 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. Atfirstitwasreally expansive to sequencinga DNAmolecule, efficiency
wasnotreally good andit took long time tosequencingamolecule.
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, 100-150bp reads 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, there will be
a fluorescent signal indicating
the base that has been added.

18. Theslide isthenpreparedforthe next cycle.The terminators are
removed, allowing thenextbaseto be added,andthe fluorescent
signal is removed, preventingthe signal from contaminatingthenext
image.
Theprocess isrepeated, addingone nucleotideata time andimaging
inbetween.

19. Computersare thenused to detectthebaseateachsite in eachimage
andthese are usedto constructa sequence.
All of thesequence readswill be the same length,as thereadlength
dependson thenumberof cyclescarriedout.

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-specifc primers.
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
isadded 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 Gs will
beadded.

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 NTPmix is washedaway.The next NTPmix is now addedandthe
processrepeated, cyclingthroughthefourNTPs.

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. ThedNTPsarewashedaway,and theprocessis repeated cycling
throughthedifferent dNTPspecies.

28. ThepH change,if any,is used to determine howmanybases (if any)
were addedwitheach 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. GridION Nanopore

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

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

39. Sources
Wong, L.-J. C., 2013. Next Generation Sequencing: Translation to Clinical Diagnostics.
1st ed. NewYork: springer
Jay Shendure & Hanlee Ji, 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. Thankyouforyourattention