The document discusses sequencing methods in biology, focusing on traditional methods like Maxam-Gilbert and Sanger sequencing, as well as next-generation sequencing (NGS) and its advantages such as speed and cost-effectiveness. NGS technologies enable the high-throughput sequencing of millions of DNA fragments simultaneously, using techniques like emulsion PCR and cyclic reversible termination. It also outlines various NGS platforms, applications, and bioinformatics challenges related to genome alignment and assembly.

1. 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.

2. 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.

3. 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.

4. NEXT GENERATION SEQUENCING
•Also known as
▫High throughput sequencing or
▫ultra-deep sequencing or
▫massively parallel sequencing.

5. 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.

6. Major Platforms for NGS
•454 ( By Roche)
•SOLiD (By Applied Biosystems)
•Solexa (By Illumina)

7. • 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.

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

9. 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

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

11. 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.

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

13. 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)

14. PCR amplification
Pico Titre Tube
• Adapter containing the
universal priming site
are ligated to target
ends
• Same primer can be
used for amplification

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

16. Nucleotide addition
Output

18. 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.

19. 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
• Alignment of sequence reads to a
reference
BLAST doesn’t blast here
Short read aligners side-lines BLAST
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. • Further Readings
1. Branton, D. et al. The potential and challenges
of nanopore sequencing. Nature Biotech. 26
1146–1153 (2008).
2. Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a
revolutionary tool for transcriptomics. Nature
Rev. Genet. 10, 57–63 (2009).
3. Petrosino, J. F., Highlander, S., Luna, R.
A., Gibbs, R. A. & Versalovic, J. Metagenomic
pyrosequencing and microbial identification.
Clin. Chem. 55, 856–866 (2009).
4. Park, P. J. ChIP–seq: advantages and challenges
of a maturing technology. Nature Rev. Genet.
10, 669–680 (2009).