a fundamentally different approach to sequencing triggering numerous ground-breaking discoveries and igniting a revolution in genomic science

1. NEXT GENERATION
SEQUENCING
A FUNDAMENTALLY DIFFERENT APPROACH TO SEQUENCING
TRIGGERING NUMEROUS GROUND-BREAKING DISCOVERIES AND
IGNITING A REVOLUTION IN GENOMIC SCIENCE.
Presented by –
VISHAL PANDEY
17412GPB017
M.Sc. GPB, Sem-1

2. What is sequencing ?
 Deciphering the code hidden in biological sequences like DNA,
polypeptides etc.
 Method and technologies that enables us to determine the order
of nucleotides and amino acids in DNA and Polypeptide
respectively.

3. Traditional methods of
Sequencing and its limitations
Maxam-Gilbert Method
 Use of radioactive labels.
Sanger Method
 It utilize the fluorescent dye for labeling.
 Separation of extended fragments of DNA with the addition of di-
deoxynucleotides (lack a 3’-OH group) Thus, chain termination.
Limitation
 Slow
 High cost per run.

4. Automated Sanger method
1. Bacterial cloning or PCR
template purification
2. Labelling of DNA
fragments using the chain
termination method with
energy transfer
3. Dye-labelled di-de
oxynucleotides and a DNA
polymerase
4. Capillary electrophoresis
5. Fluorescence detection
that provides four-colour
plots to reveal the DNA
sequence.

5. NEXT GENERATION
SEQUENCING
Also known as -
 High throughput sequencing or
 ultra-deep sequencing or
 massively parallel sequencing.

6. What is next generation
sequencing ?
 Automated Sanger method (1st generation)
 Technologies developed after that are known as next
generation sequencing.
 NGS enables the sequencing of biological codes at a
very rapid pace with low cost per operation.
 This is the primary advantage over conventional
methods.
 For example Billions of short reads can be sequenced in
one operation.

7. Major Platforms for NGS
 454 ( By Roche)
 SOLiD (By Applied Biosystems)
 Solexa (By Illumina)

8.  Above mentioned platform varies in strategies,
application and type of data generated.
 However, all technologies are common in -
 That they generate sequences on an unprecedented
scale
 DNA cloning is not required
 and very low operation cost.

9. What NGS Consists of
Next generation technologies for sequencing is
combination of strategies for
 template preparation
 sequencing and imaging
 genome alignment
 assembly methods

10. Template preparation
As even most sensitive imaging technique is not able to
detect single molecule, amplification of templates is
inevitable.
 Clonally amplified templates
 By emulsion PCR (emPCR) e.g. 454 and SOLiD
 Solid phase amplification e.g. illumina
 Single-molecule templates

11. Template preparation:
Traditional vs NGS
 Immobilization of templates fragments over
bead /glass plate allows billions of the
sequencing reaction run simultaneously

12. sequencing and imaging
 Sequencing
 cyclic reversible termination(CRT) e.g. illumina/solexa
 single-nucleotide addition (SNA) e.g. 454/roche
 real-time sequencing: R&D going on (pacific Bioscience)
 Sequencing by ligation (SBL) e.g. SOLiD
 Imaging
 measuring bioluminescent signals
 four-colour imaging of single molecular events e.g.
illumina/solexa.

13. Genome alignment and
assembly
After NGS reads have been generated, they
are aligned to either
 a known reference sequence
or
 assembled de novo

14. 454 (Pyrosequencing)
 DNA is fragmented, joined to
adapters at either end of the
fragmented DNA
 amplified in an emulsion PCR
(includes agarose bead with
complimentary adaptors to
fragmented DNA)
 PCR amplified allowing up to 1
million identical fragments around
one bead and finally dropped into a
PicoTitreTube (PTT)

15. PCR amplification Pico Titre
Tube
 Adapter containing the universal
priming site are ligated to target ends
 Same primer can be used for
amplification
PCR Amplification
PICO Titre Tube

16. In Pico titre tube reaction
of fluorescence occurs
with the addition of
nucleotides Nucleotide
addition

17. Nucleotide addition Output

19. SOLiD (support oligonucleotide
ligation detection)
 Sequencing by Oligo/Ligation and
Detection.
 Steps
 Library Preparation
 two types of libraries sequencing-
fragment or mate-paired are prepared.
 Emulsion PCR/Bead Enrichment
 amplification of template fragments is
done in same manner as 454.
 Bead Deposition
 Deposit 3’ modified beads onto a glass
slide.

20. Sequencing by Ligation
 Primers hybridize to the P1 adapter
sequence on the templated beads
 The method uses two-base- encoded
probes(4 probes), which has the primary
advantage of improved accuracy.
 Multiple cycles of ligation, detection and
cleavage are performed.
 Extension product is removed and the
template is reset with a primer
complementary to the n-1 position for a
second round of ligation cycles.

21. Illumina
 Breaking up DNA
 Adding adaptors, but in this case
attach not to a bead but to a slide
 Fold-back PCR is then used to
amplify the fragmented DNA into a
cluster

22. Sequential
addition of
nucleotides
are added
using a
polymerase

23. NGS and Bioinformatics
 Software
 Bowtie
 MAQ
 BWA

24.  Above strategy works if reference genome exist.
 de novo assembly from paired or unpaired reads
 base-calling and/or polymorphism detection
 structural variant detection
 genome browsing.

25. Application of NGS
 Variants discovery in targeted region or whole genome by re-
sequencing
 Reassembling genome of lower organism by de novo method.
 Cost-effective sequencing of complex samples at remarkable scale
and speed.
 Sequencing entire transcriptome.
 In Meta genomics : Sequencing genome of entire biological
communities
 Replacing ChIP-on-chip with ChIP-seq in case of multicellular
eukaryotes.
 Personalized genome for personalized medicine

26. Thank you