classic and next -gen sequencing...

Classic and NextClassic and Next--Gen Gen

Sequencing TechnologiesSequencing Technologies

6th July 2009

David Miller

Australian Genome Research Facility

Overview of PresentationOverview of Presentation

MilestonesMilestones1953 Discovery of the structure of the DNA double helix.

1972 Development of recombinant DNA technology, which permits isolation of defined fragments of DNA; prior to this, the only accessible samples for sequencing were from bacteriophage or virus DNA.

1975 The first complete DNA genome to be sequenced is that of bacteriophage φX174

1977 Allan Maxam and Walter Gilbert publish "DNA sequencing by chemical degradation".[3] Frederick Sanger, independently, publishes "DNA sequencing by enzymatic synthesis".

1980 Frederick Sanger and Walter Gilbert receive the Nobel Prize in Chemistry

1984 Medical Research Council scientists decipher the complete DNA sequence of the Epstein-Barr virus, 170 kb.

1986 Leroy E. Hood's laboratory at the California Institute of Technology and Smith announce the first semi-automated DNA sequencing machine.

1987 Applied Biosystems markets first automated sequencing machine, the model

ABI 370.

1990 The U.S. National Institutes of Health (NIH) begins large-scale sequencing trials on Mycoplasma capricolum, Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae (at 75 cents (US)/base).

1995 Richard Mathies et al. publish dye-based sequencing.

1998 Phil Green and Brent Ewing of the University of Washington publish “phred” for sequencer data analysis.

History of DNA SequencingHistory of DNA Sequencing

Frederick SangerOnly person to receive two Noble prizes in Chemistry.

One of only four people ever to receive two Nobel prizes.

Only living person to receive two Nobel prizes.

• Brief Overview

• 15 year project began in 1990

• US $3 Billion dollar project

• 2000 first draft genome was announced

• 2003 complete genome announced

• 2006 final chromosome published in nature

Human Genome ProjectHuman Genome Project

Human Genome ProjectHuman Genome Project

Project Goals• identify all the approximately 20,000-25,000 genes in human

DNA,

• determine the sequences of the 3 billion chemical base pairs that make up human DNA,

• store this information in databases,

• improve tools for data analysis,

• transfer related technologies to the private sector, and

• address the ethical, legal, and social issues (ELSI) that may arise from the project.

Human Genome Project Human Genome Project Goals and Completion Dates

Area HGP Goal Standard Achieved Date Achieved

Genetic Map 2- to 5-cM resolution map (600 – 1,500 markers)

1-cM resolution map (3,000 markers)

September 1994

Physical Map 30,000 STSs 52,000 STSs October 1998

DNA Sequence 95% of gene-containing part of human sequence finished

to 99.99% accuracy

99% of gene-containing part of human sequence finished to

99.99% accuracy

April 2003

Capacity and Cost of Finished Sequence

Sequence 500 Mb/year at < $0.25 per finished base

Sequence >1,400Mb/year at <$0.09 per finished

base

November 2002

Human Sequence Variation 100,000 mapped human SNPs

3.7 million mapped human SNPs February 2003

Gene Identification Full-length human cDNAs 15,000 full-length human cDNAs March 2003

Model Organisms Complete genome sequences of

E. coli, S. cerevisiae, C. elegans,D. melanogaster

Finished genome sequences of E. coli,S. cerevisiae, C. elegans,D. melanogaster, plus whole-

genome drafts of several others, including C. briggsae, D.

pseudoobscura, mouse and rat

April 2003

Shotgun Sequencing ApproachShotgun Sequencing Approach

Classic Sequencing Classic Sequencing

TechnologiesTechnologies

1977

Allan Maxam and Walter Gilbert publish "DNA sequencing by chemical degradation".[3] Frederick Sanger, independently, publishes "DNA sequencing by enzymatic synthesis".

Both methods generate fragment pools which are resolved via electrophoresis with a resolution of 1 BP.

1986 4 Reactions to 1 Lane1986 4 Reactions to 1 Lane

Sequencing Reaction Products Progression of Sequencing Reaction

Classic Sequencing Classic Sequencing


ABI377ABI377

Capillary Based ElectrophoresisCapillary Based Electrophoresis

Raw DataRaw Data

ElectropherogramsElectropherograms

ABI3700ABI3700

MegaBACEMegaBACE 10001000

ABI3730xlABI3730xl

Sanger SequencingSanger Sequencing

Maximum yield / days <3,000,000bp <0.1% of the human genome 1000 days of sequencing for a 1 fold coverage.....

NextNext--Gen Sequencing Gen Sequencing




• Three platforms, three technologies

• All massively parallel sequencing

• Read lengths vary from ~36bp to >400bp

• Read numbers vary from ~ 1 Million to >150 Million per run

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

3.50E+06

4.00E+06

4.50E+06

3730- 96

3730- 384

GS-20

GS-FLX

GS-FLXTI

GAII1x 18

GAII1x 26

GAII1x 35

GAII2x 35

GAIIx2x 35

GAII2x 50

GAIIx2x 50

GAII2x 75

GAIIx2x 75



Instrument Run Type

Raw

Data

Sto

rage (

Mb)



Roche GS-FLX Illumina GAII ABI SOLiDRoche GS-FLX

Roche GSRoche GS--FlxFlx

WorkflowWorkflow

• Library Preparation

• emPCR Setup

• emPCR Amplification

• Pyrosequencing

• Data Analysis

• Sample Fragmentation

Sample Fragmentation and Sample Fragmentation and

Library GenerationLibrary Generation

emPCRemPCREmulsion PCR is a method of clonal amplification which allows

for millions of unique PCRs to be performed at once through the generation of micro-reactors.

emPCR

The Water-in-Oil-Emulsion

PyrosequencingPyrosequencing

Data AnalysisData Analysis

Raw Image Files

Platform UpdatesPlatform Updates

Maximum yield / days 1,000,000,000bp 30% of the human genome ~3 days of sequencing for a 1 fold coverage.....

IlluminaIllumina GAIIGAII

IlluminaIllumina Library PreparationLibrary Preparation

Stylised graphic of the Paired-End library preparation taken from the Paired-End Protocol

DNA(0.1-1.0 ug)

Sample preparation Cluster growth

5’

5’3’

G

T

C

A

G

T

C

A

G

T

C

A

C

A

G

TC

A

T

C

A

C

C

TAG

CG

TA

GT

1 2 3 7 8 94 5 6

Image acquisition Base calling

T G C T A C G A T …

Sequencing

IlluminaIllumina Sequencing TechnologySequencing TechnologyRobust Reversible Terminator Chemistry Foundation

IlluminaIllumina GAII Development GAII Development

2009 Roadmap2009 Roadmap

SCS 2.4 and Pipeline 1.4

• Two recent software updates from Illumina

• Huge impact on data yields by not only increasing the number of clusters detectable, but also the number of clusters that pass quality filtering

• Originally aimed for ~50 Million reads / run

• Now aiming for ~150 Million reads / run

Platform UpdatesPlatform Updates

Maximum yield / days 2,000,000,000bp 60% of the human genome ~1.5 days of sequencing for a 1 fold coverage.....

ABI ABI SOLiDSOLiD

ABI ABI ColorSpaceColorSpace

ABI ABI SOLiDSOLiD

emPCRemPCR and Enrichmentand Enrichment

Bead DepositionBead Deposition

3’ Modification allows covalent bonding to the slide surface

Sequencing by LigationSequencing by Ligation

Base InterrogationsBase Interrogations

Genome Web

Summary

• Both SOLiD and GS-FLX use emPCR

• Both GS-FLX and GAII sequence by synthesis

• GAII uses “cluster generation” similar to the polony approach

• SOLiD sequencing by ligation

• GS-FLX provides ~100x decrease in costs compared to Sanger Sequencing

• GAII and SOLiD ~10x decrease in costs over GS-FLX (though this is probably increasing given the huge increase in output)

ApplicationsApplications

Targeted Enrichment

• Sequencing costs have shifted significantly from sequencing to up-front sample preparation, libraries, amplification…

• Recent applications from Roche Nimblegen and Agilent Technologies have allowed for sequence capture through the use of microarrays

• Resulted in two products, Nimblgen Sequence Capture Arrays and Agilent SureSelect

SureSelect In-Solution Capture

Nimblegen Sequence Capture Arrays

PETPET--SEQSEQ

PET-SEQ

P1 Tag1

Mate-Pair

P2Tag2InternalAdapter

DeletionsInsertions

Tag Reads

Reference

Inversions

Tag Reads

Reference

NextNext--NextNext--Gen Sequencing Gen Sequencing


Websites of NoteWebsites of Note

http://www.illumina.com/pages.ilmn?ID=203

http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/index.htm

http://www.454.com/index.asp

http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml

http://www.helicosbio.com/

http://www.pacificbiosciences.com/

http://www.nanoporetech.com/sequences/

THANKSTHANKS

6th July 2009

David Miller

Australian Genome Research Facility

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