DNA-Sequencing

Technologies & Devices

ABI 3730xl 4/2004 & 6/2006

1 Mb/day, 850 nt reads

Roche/454 GS FLX 12/2006

Illumina/Solexa GAIIx 12/2008 & 11/2009

80 Gb/14d, 2x150 nt reads

HiSeq 2000 12/2010

2x300 Gb/10d, 2x100 nt reads

Genome analysis DNA sequencing platforms

2x150 nt reads

2 Mb/day, 550 nt reads

800 Mb/23h, 800 nt reads

Moore’s law Next Generation Sequencing - a game changer

http://ivory.idyll.org/permanent/lstein-ngs-capacity.png

NGS Terminology

2nd generation = massively parallel 3rd generation = true single-molecule

1st Generation

Maxam-Gilbert . Sanger . Pyro

Maxam-Gilbert sequencing Chemical modifications, radioactive *1977

Sanger sequencing Dideoxy chain termination, radioactive *1977

Fred Sanger Nobel prices 1958, 1980

Sanger sequencing Fluorescence labeling

Sanger sequencing Dye primer

Sanger sequencing Dye terminator

DP

DT

Sanger sequencing Dye primer vs. terminator

PNAS 92:6339 (1995)

Thermo-Sequenase = Taq polymerase Phe>Thy mutated

Sanger sequencing Engineered polymerase

Sanger sequencing Cycle sequencing

PPi

ATP

Pyrosequencing

2nd Generation

Roche/454. Illumina/Solexa . ABI/Solid ABI/IonTorrent . Complete Genomics

A Revolutionary Method for Rapid Whole Genome Sequencing and Assembly And more……..

454 Genome Sequencer GS20/FLX/FLX+

454 Nature, July 31 2005

• 25 Mb • 99% accuracy • 4h

• de novo assembly Mycoplasma genitalium (0.58 Mb): 96% coverage, 99,96% accuracy

• 100x currant Sanger technology

454 Nature, July 31 2005

Roche/454 Workflow

dsDNA fragments

P A B Ligation

Fill in

Capture on SA-Beads & Wash Alkaline Elution

A B

P Bio

Bio

Bio

Bio

Bio

Roche/454 Preparation of A/B-fragments for sequencing

Roche/454 emPCR

Roche/454 Sample loading

Roche/454 Flowgram

GC normal high-cloned coverage

G+C

%

green - 454 reads red - Sanger reads black - GC content

GC poor low-cloned coverage

Roche/454 No cloning bias

Background subtracted

Primer sequence

C to T ratio: 1/500

T only

Primer sequence

0.16%

Mut

atio

n Fr

eq.

Mut

atio

n Fr

eq.

Cov

erag

e C

over

age

Roche/454 Deep sequencing – rare mutation detection

Roche/454 Read length

Roche/454 Read length

Advanced genetic analysis

one billion bases at a time

Solexa Technology

• Clonal Singe Molecule Array TM

• Sequencing By Synthesis (SBS)

• reversible fluorescent labels

• reversible 3’OH blocking

Illumina/Solexa Linker ligation

Illumina/Solexa Solid-phase clonal single molecule PCR

“Bridge amplification”

100μm

colony of ≈1000 single-stranded DNA templates

Single well of 454 Life Sciences PicoTiterPlateTM (to same scale)

Illumina/Solexa Solid-phase clonal single molecule PCR

Illumina/Solexa Sequencing by synthesis (SBS)

removal of fluorescent labels & 3’OH blocking *

100μm

colony of ≈1000 single-stranded DNA templates

Illumina/Solexa Sequencing by synthesis (SBS)

1 2

Illumina/Solexa Assembly - Mapping

read length: 25-36 … 76 … 102 … 150 … 250 nt

Illumina/Solexa Paired ends & Mate pairs

Illumina/Solexa Multiplexing

Illumina/Solexa Data output

Agencourt

ABI/SOLID Bead deposition

ABI/SOLID Probes

16

ABI/SOLID 2-base encoding

16 dinucleotides / 4 dyes = 4 dinucleotides/dye

ABI/SOLID 4-color ligation

ABI/SOLID 4-color ligation

ABI/SOLID Detection

ABI/SOLID Cleavage

ABI/SOLID 2nd cycle ligation

ABI/SOLID 2nd cycle detection

ABI/SOLID 2nd cycle cleavage

+ additional 3 ligation cyles

ABI/SOLID Reset after 5th cycle

ABI/SOLID 6th cycle ligation

ABI/SOLID Cycling scheme

ABI/SOLID 2-base encoding

ABI/SOLID Paired ends – two sequences

ABI/SOLID Performance

www.completegenomics.com

Complete Genomics In-house technology

• self-assembling DNA nanoballs (DNB) • patterned nanoarrays • combinatorial probe anchor ligation (cPAL)

Drmanac et al. Science 327:78 (2009)

Complete Genomics Performance

• launched by mid-2010

• 2010: ~600 human genomes • 2011: ~4,000 genomes

• end 2011: 800-1200 genomes/month • mid 2012: new machines with 6 genomes/day • end 2012: <3,000 $/genome

www.iontorrent.com

PGM - Personal Genome Machine

ABI/Ion Torrent The chip is the machine

ionogram www.iontorrent.com

ABI/Ion Torrent Read length

ABI/Ion Torrent Data output

ABI/Ion Torrent Data output

Nature 475:348 (2011)

ABI/Ion Torrent ‘Post-light’ sequencing of Gordon Moore

2nd generation sequencing Applications

• Re-sequencing

• RNA-seq

• DigiTag-seq

• miRNA-seq

• ChIP-seq

• Ribosome-footprinting

• …

3rd Generation

Helicos. Pacific Biosciences VisigGen . Nanopore(s)

www.helicosbio.com

Helicos First true single molecule sequencing (tSMS)

www.helicosbio.com

Helicos DRS

Nature 461:814 (2009)

www.pacificbiosciences.com

et al. : 2

Pacific Biosciences Technologogies

Pacific Biosciences Fluorescent lable

www.pacificbiosciences.com

Pacific Biosciences Immobilised polymerase

long sequences

www.pacificbiosciences.com

Pacific Biosciences Zero-mode waveguide chip

www.pacificbiosciences.com

Tex

Pacific Biosciences Real-time DNA methylation detection

Nature Methods 7:461 (2010)

Pacific Biosciences Epigenetics

www.pacificbiosciences.com

Pacific Biosciences Direct RNA sequencing

www.pacificbiosciences.com

Pacific Biosciences Translation

www.pacificbiosciences.com

VisiGen Fluorescence resonance energy transfer (FRET)

F-donor : polymerase F-acceptor: γ-phosphate of dNTP

1Mb/s >1 human genome/day

visigenbio.com

Nanopore Ion current

Messwert Strom von K+-Ionen, die der DNA beim Porendurchtritt entgegen fließen

www.mcb.harvard.edu/branton/nanopore.html

IBM Waver pore

genome.fli-leibniz.de Lectures