next generation sequencing
DESCRIPTION
Next generation SequencingTRANSCRIPT
NEXT GENERATION SEQUENCING METHODS
Course teacher ByDr. N. Senthil Nidhi Singh (09-607-
010)
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.
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,
SANGER DDNTP CHAIN TERMINATION SEQUENCING
Daniel, 2009
MAXAM-GILBERT METHOD
COUNTRIES CONTRIBUTION TO SEQUENCING
2010
MAJOR SEQUENCING CENTERS
ADVANCEMENT IN SEQUENCING
2010
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
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
•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
SEQ-BY-SYNTH 454 LIFE SCIENCES PLATFORM
Wilhelm , 2009
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
SEQUENCING BY LIGATIONDNA fragment ligated to 1 µm Polystyrene/magnetic bead.
Amplification by Emulsion PCR
Universal sequencing primer complementary to adaptor
Shendure et al, 2008
SOLiD: Sequencing ligation cycles
Shendure et al, 2008
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
HELISCOPE(TRUE SINGLE MOLECULE SEQUENCING)
Developed by Helicos Biosciences in 2007.
No Amplification needed
Single molecule detection
Homopolymer stretches(solved)
Expensive Detection
Wilhelm , 2009
Wilhelm, 2009
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
• 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
Shendure et al, 2008
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
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
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
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
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.
APPLICATION
Avak et al., 2008
COMPANIES DETAIL
Elim Biopharmaceuticals, Inc
leading , in the San Francisco Bay AreaElim 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.
DISCUSSION
THANK YOU