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ADVANCES IN SEQUENCING

TECHNOLOGY

Dr.M.Vinoth
Animal biotechnology
Adapted from Eric Green, NIH; Adapted from Messing & Llaca, PNAS (1998)
History of DNA Sequencing

1870 Miescher: Discovers DNA

Avery: Proposes DNA as ‘Genetic Material’


Efficiency 1940
Watson & Crick: Double Helix Structure of DNA
(bp/person/year)

1 1953 Holley: Sequences Yeast tRNAAla

1 1965 Wu: Sequences λ Cohesive End DNA


5
15
0 1970
1,50 Messing: M13 Cloning
0
1977
15,0 Sanger: Dideoxy Chain Termination
00 Gilbert: Chemical Degradation
25,0 1980
00 Hood et al.: Partial Automation

50,0 1986
00
• Cycle Sequencing
200,0 1990 • Improved Sequencing Enzymes
00 • Improved Fluorescent Detection Schemes
50,000,00
0 2002
• Next Generation Sequencing
•Improved enzymes and chemistry
100,000,000,000 2009
•New image processing
What is need of
sequencing ?
• Fundamental requirement in biotech
• Disease genotyping-identify specific genetic
diseae(birth itself)-can give gene therapy
• Organism genome sequencing-screening and
further study about organism
• Research-primer designing, in genomics,
transcriptomics, and epigenomics etc
• Diagnostic tests,rDNA tech
Basics of the “old” technology

• Maxam and Gilbert –chemical digestion


• Sanger’s method-chain termination method
• Primer walking,chromosome walking,jumping
• Automated DNA sequencer-florescent tags in ddNTP
• HGP-2000-complete sequence
• 15 years
• $ 3 billion
ADVANCE TECHNOLOGY
• Pyrosequcing
• Nanophore sequencing
• Next-gen sequencing(Massively parallel
sequencing technology)
• VisiGen's
• illumina (Solexa)
• Applied biosystems(SOLiD)
• 454-life science
• Helicos
• Pacific-bioscience
PYROSEQUENCING
• It is based on pyrophosphate release
during DNA synthesis
• Ppi converted ATP by ATP sulfurylase
• This ATP give energy to luciferase
• Luciferase- oxidize luciferin and
generate light
• Before next step apyrase degrade
the unincorporated nucleotides
• Now add one known nucleotide
• If it attach to template- ppi release
• It generate light
• light detected by charge coupled
device camera (CCD-camera)
• If added nucleotide is not
complementary to template, Light is
not generate
Roche / 454 : GS FLX

• Also known as “pyrosequencing”


• 500 million bp/run
• 400-500 bp/read
Roche / 454 : GS FLX
Roche / 454 : GS FLX
Roche / 454 : GS FLX
disadvantage
• the number of repetitive bases,
erroneous base calls can be a
problem with homopolymers.
• Another disadvantage of 454
sequencing is that while it is cheaper
and faster per base, each run is quite
expensive
Polymerase sequencing
• VisiGen Biotechnologies is developing a
revolutionary approach for DNA sequencing that
will enable rapid, inexpensive whole genome
sequencing
• Fluorescently tagged polymerase and
Fluorescently modified nucleotide is used
• In this each nucleotide have specific colour coded
with florescent material
• During extension nucleotide incorporated
into growing polymer
• So base specific signature is emitted
• Achieve a sequencing rate of
1Mb/sec/machine
• Employs direct analysis and
eliminates the need for cloning or
amplification
• total cost of over $300 million to
sequence the human genome
Illumina (Solexa)
• Illumina sequencing technology
leverages clonal array formation
• proprietary reversible terminator
technology for rapid and accurate
large-scale sequencing
• all four nucleotides are added at each
cycle so accuracy more than
other
• 300Mb per day
Illumina (Solexa)
Adapted from Richard Wilson, School of Medicine, Washington University, “Sequencing the Cancer Genome” http://tinyurl.com/5f3alk

Solexa-based Whole Genome Sequencing


Illumina (Solexa)
From Debbie Nickerson, Department of Genome Sciences, University of Washington, http://tinyurl.com/6zbzh4
Applied Biosystems
SOLiDTM
Sequencer
• The SOLiD platform uses an adapter-
ligated fragment library similar to
those of the other next-generation
platforms
• uses an emulsion PCR approach with
small magnetic beads to amplify the
fragments for sequencing.
• SOLiD uses DNA ligase
SOLiD color space
SOLiD color space
SOLiD color space
SOLiD color space
SOLiD color space
SOLiD color space
Next-gen
From John McPherson, OICR

sequencers
100 Gb
AB/SOLiDv3, Illumina/GAII
short-read sequencers
(10+Gb in 50-100 bp reads,
10 Gb
$1000 ,
bases per machine run

1 Gb 454 GS FLX pyrosequencer


(100-500 Mb in 100-400 bp reads,
100 Mb

10 Mb ABI capillary sequencer


(0.04-0.08 Mb in 450-800 bp reads,
1 Mb

10 bp 100 bp 1,000 bp
read length
Helicosbio
• Helicos True Single Molecule Sequencing
(tSMS)™
• Eliminating the need for amplification and
its associated biases and error
• billions of single molecules of sample DNA
are captured on an application-specific
proprietary surface.
• Polymerase and one fluorescently labeled
nucleotide (C, G, A or T) are added.
• The polymerase catalyzes the
sequence-specific incorporation of
fluorescent nucleotides into templates.
• After a wash step,the incorporated
nucleotides are imaged and their
positions recorded.
• Then fluorescent group is removed.
Then next nucleotide incorporated .
• images billions of single molecules
per run and produces over one
gigabase of usable sequence data
per day
• throughput performance- 100X
greater than Sanger methods
• Whole genome-around $1000
pacificbiosciences
• Instead of inspecting DNA copies after
polymerase
• Single Molecule Real-Time (SMRT)
sequencing watches the enzyme in real
time
• DNA Polymerase attach on the surfase of
tiny well
• Every nucleotide fluorescently label with
different colour when it incorporate emit
different wave length light
pacificbiosciences
pacificbiosciences
• Long read,fast results low overall
cost
• now they sequencing genome for
$1000
$100 genome
• could enable
sequencing in 15 minutes in
2013.
REFERENCE

• http://454.com/
• http://illumina.com/
• http://appliedbiosystems.com/

• http://pacificbiosciences.com/
• http://helicosbio.com

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