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