Next generation sequencing techniques have revolutionized DNA sequencing by increasing throughput and decreasing costs compared to previous methods like Sanger sequencing. Some key next generation sequencing methods include 454 sequencing (pyrosequencing), ABI Solid sequencing (sequencing by ligation), Illumina/Solexa sequencing (sequencing by synthesis), and nanopore sequencing. These new techniques allow for faster and cheaper large-scale sequencing and have enabled applications like whole genome sequencing.

1. NEXT GENERATION SEQUENCING
METHODS
Course teacher By
Dr. N. Senthil Nidhi Singh
(09-607-010)

2. WHAT IS SEQUENCING?
 “Sequencing” means finding the order of nucleotides
on a piece of DNA .
 Nucleotide order determines amino acid order, and by
extension, protein structure and function.
 An alteration in a DNA sequence can lead to an altered
or non functional protein, and hence to a harmful effect
in a plant or animal.

3.  The sequencing of the reference human genome was the
capstone for many years of hard work spent developing high-
throughput.
 Scenario is rapidly changing owing to the invention and
commercial introduction several revolutionary approaches to
DNA sequencing, the so-called next-generation sequencing
technologies
 Major impact on our ability to explore and answer genome-
wide biological questions.
 Not only changing our genome sequencing approaches and the
associated timelines and costs, but also accelerating and
altering a wide variety of types of biological inquiry that have
historically used a sequencing-based readout,

4. SANGER DDNTP CHAIN TERMINATION
SEQUENCING
Daniel, 2009

5. MAXAM-GILBERT METHOD

6. COUNTRIES CONTRIBUTION TO SEQUENCING
2010

7. MAJOR SEQUENCING CENTERS

8. ADVANCEMENT IN SEQUENCING
2010

9. NEXT GENERATION SEQUENCING TECHNIQUES
 454 Sequencing (Pyro sequencing)
 ABI Solid (Sequencing By Ligation)
 Helicos (True Single Molecule Sequencing)
 Nanopore (Single Base Resolution)
 Illumina /Solexa: Genetic Analyzer

10. ROCHE (454) GS FLX
 In 2005, Developed by 454 Life Sciences.
 Follows Sequencing-By-Synthesis chemistry.
 Initially with 96 sample capacity in parallel in a microtiter
plate.
 Now Picotiter plates & Streptavidin beads are used
allowing about 1 million seq reads.
 GS FLX Titanium series sequence 400-600 million bases
per 10-hour run.
Wilhelm , 2009

11. •454 is based pyrosequencing on the generation of light
signal through release of pyrophosphate (PPi) on
nucleotide addition.
DNAn + dNTP  DNAn+1 + PPI
•PPi is used to generate ATP from adenosine
phosphosulfate (APS).
APS + PPI  ATP
•ATP and luciferase generate light by conversion of
luciferin to oxyluciferin.
Principle
Wilhelm ,2009

12. SEQ-BY-SYNTH 454 LIFE SCIENCES PLATFORM
Wilhelm , 2009

13. ABI SOLID
 Sequencing by Oligo Ligation and
Detection.
 Also called as ‘2 base encoding’.
 Developed by Applied Biosystems in
Autumn 2007.
 In 2008,updated version ABI SOLid
2.0 platform is commercialized.
 Polystyrene beads are used.
Shendure et al, 2008

14. SEQUENCING BY LIGATION
DNA fragment ligated to 1 µm
Polystyrene/magnetic bead.
Amplification by Emulsion PCR
Universal sequencing primer complementary to
adaptor
Shendure et al, 2008

15. SOLiD: Sequencing ligation
cycles
Shendure et al, 2008

16. Decoding of paired bases is done
by determing the overlapping
bases from each pair.
Sequences are Computated and
sequence result is displayed
Shendure et al, 2008

17. HELISCOPE
(TRUE SINGLE MOLECULE SEQUENCING)
 Developed by Helicos Biosciences in 2007.
 No Amplification needed
 Single molecule detection
 Homopolymer stretches(solved)
 Expensive Detection
Wilhelm , 2009

18. Wilhelm, 2009

19. ILLUMINA GENOME ANALYZER
•single molecule amplification
•starts with a Illumina-specific adapter
library
•performed by an automated device called
Cluster Station
•uses DNA polymerase to produce multiple
DNAcopies,
•utilizes a sequencing by synthesis approach
•the nucleotides carry a base-unique
fluorescent label
•the 3-OH group is chemicall blocked such
that each incorporation isa unique event
Shendure et al, 2008

20. •Imaging step is followed with each base incorporation
•each flow cell lane is imaged in three 100-tile
segmentby the instrument optics at a cluster densitper
tile of 30,000
• After each imaging step the 3-OH blocking group is chemically
remove
• quality checking pipeline evaluates the Illumina data from
each run
• A base-calling algorithm assigns sequences and associated
quality values to each read

21. Shendure et al, 2008

22. NANO-PORE DNA SEQUENCING
 Nanopore is a nanoscale pore in an
electrically insulating membrane
- a pore in a solid-state membrane (most
commonly Si3N4)
 Nanofabricated pores that are contained in
biological or inorganic membranes might
allow for the sequencing of individual
molecules of DNA.
Eugene, 2005

23.  As the molecule
translocates across the
membrane , the identity of
individual nucleotides in
the molecule of DNA is
determined using ionic
current or transverse
current .
Eugene, 2005

24.  DNA translocates across the nanopore and
produces a change in the transverse current
between two electrodes that are on either
side of the nanopore.
 Nanopores can also operate using changes in
ionic current, that is, the flow of positive ions
in the opposite direction to the negatively
charged DNA translocating the nanopore.
Eugene, 2005

25. Sequencing
Chemistry
Read
Length
Cost per
instrument
Total
output
TIME/run Accuracy
Sanger Sequencing-
By-Synthesis
800 bp -- 700 Mb-1
Gb
-- 99 %
454 GS Sequencing -
by-Synthesis
300-800
bp
$500,000 4 Gb 7 hr 99 %
ABI
SoLiD
Sequencing-
by-Ligation
25-35 bp $591,000 3-10 Gb 4.5 Days 99.94 %
Heliscope True Single
Molecule
Sequencing
30-55 bp $1,350,000 21-35 Gb 14 Days 99.99 %
Nanopore Single
Molecule
Resolution
5.4 kb -- 35-76 Gb 11 hr 99.9 %
COMPARISION OF SEQUENCING METHODS
William Stafford Noble , 2010

26. CONCLUSION
•The new method are fast as it takes about 1
day to sequence 100 million bases.
• Inexpensive due to lost per base sequencing.
• Accurate by using a sequencing array and high
accuracy can be achieved by analyzing several
time series.

27. APPLICATION
Avak et al., 2008

28. COMPANIES DETAIL
Elim Biopharmaceuticals,
Inc
leading , in the San Francisco Bay Area
Elim has pioneered many new features in DNA
sequencing services
Bioaxis DNA Research
Centre
private limited , top level CRO in life sciences ,
vast range of services in Biotechnology,
Bioinformatics and Clinical Research
NanoBioServices Chandigarh, India NanoBioServices is a
contract research and contract manufacturing
organization
Xcelris Gujarat, India Xcelris provides bio-analytical
services., includes SNP analysis, m-RNA
profiling
Genewiz, Inc. South Plainfield, New Jersey Genewiz, Inc.
is a contract research organization specializing
in DNA sequencing, molecular biology
DNA Analysis, LLC Cincinnati, Ohio DNA Analysis, LLC provides
complete sequencing and fragment analysis
service including analysis
MWG Biotech Pvt. Ltd Karnataka, India MWG Biotech Pvt. Ltd.
Focuses on genotyping and bioinformatics.