vorles-seqtec 111206.ppt [kompatibilitätsmodus] · dna sequencing platforms 2x300 gb/10d, ......

DNA-Sequencing

Technologies & Devices

ABI 3730xl4/2004 & 6/2006

1 Mb/day, 850 nt reads

Roche/454 GS FLX12/2006

Illumina/Solexa GAIIx12/2008 & 11/2009

400 Mb/7h, 350 nt reads

80 Gb/14d, 2x150 nt reads

HiSeq 200012/2010

2x150 Gb/10d, 2x100 nt reads

Genome analysisDNA sequencing platforms

2x300 Gb/10d, 2x100 nt reads

2 Mb/day, 550 nt reads

800 Gb/23h, 800 nt reads

Moore’s lawNext Generation Sequencing - a game changer

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

NGSTerminology

2nd generation= massively parallel

3rd generation= true single-molecule

1st Generation

Maxam-Gilbert . Sanger . Pyro

Maxam-Gilbert sequencingChemical modifications, radioactive *1977

Sanger sequencingDideoxy chain termination, radioactive *1977

Fred SangerNobel prices 1958, 1980

Sanger sequencingFluorescence labeling

Sanger sequencingDye primer

Sanger sequencingDye terminator

DP

DT

Sanger sequencingDye primer vs. terminator

PNAS 92:6339 (1995)

Thermo-Sequenase = Taq polymerase Phe>Thy mutated

Sanger sequencingEngineered polymerase

Sanger sequencingCycle sequencing

PPi

ATP

Pyrosequencing

2nd Generation

454/Roche. Solexa/Illumina . Solid Ion Torrent . Complete Genomics

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

454Genome Sequencer GS20/FLX/FLX+

454Nature, 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

454Nature, July 31 2005

454Workflow

dsDNA fragments

PA BLigation

Fill in

Capture on SA-Beads & WashAlkaline Elution

A B

P Bio

Bio

Bio

Bio

Bio

454Preparation of A/B-fragments for sequencing

454emPCR

454Sample loading

454Flowgram

GC normalhigh-cloned coverage

G+C

%

green - 454 readsred - Sanger readsblack - GC content

GC poorlow-cloned coverage

454No 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

eC

over

age

454Deep sequencing – rare mutation detection

Roche/454Read length

Roche/454Read length

Advanced genetic analysis

one billion bases at a time

SolexaTechnology

• Clonal Singe Molecule Array TM

• Sequencing By Synthesis (SBS)

• reversible fluorescent labels

• reversible 3’OH blocking

SolexaLinker ligation

SolexaSolid-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)

SolexaSolid-phase clonal single molecule PCR

SolexaSequencing by synthesis (SBS)

removal of fluorescent labels & 3’OH blocking*

100μm

colony of ≈1000 single-stranded DNA templates

SolexaSequencing by synthesis (SBS)

1 2

SolexaAssembly - Mapping

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

Illumina/SolexaPaired ends & Mate pairs

Illumina/SolexaMultiplexing

Illumina/SolexaApplications

• Re-sequencing

• RNA-seq

• DigiTag-seq

• miRNA-seq

• ChIP-seq

• Ribosome-footprinting

• …

Illumina/SolexaData output

ABI AgencourtSOLID

SOLIDBead deposition

SOLIDProbes

16

SOLID2-base encoding

16 dinucleotides / 4 dyes = 4 dinucleotides/dye

SOLID4-color ligation

SOLID4-color ligation

SOLIDDetection

SOLIDCleavage

SOLID2nd cycle ligation

SOLID2nd cycle detection

SOLID2nd cycle cleavage

+ additional 3 ligation cyles

SOLIDReset after 5th cycle

SOLID6th cycle ligation

SOLIDCycling scheme

SOLID2-base encoding

SOLIDPaired ends – two sequences

SOLIDPerformance

www.completegenomics.com

Complete GenomicsIn-house technology

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

Drmanac et al. Science 327:78 (2009)

Complete GenomicsPerformance

• 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

Ion TorrentThe chip is the machine

ionogramwww.iontorrent.com

et al. : 2

Ion TorrentRead length

Ion Torrent Data output

et al. : 2

Nature 475:348 (2011)

Ion Torrent ‘Post-light’ sequencing of Gordon Moore

3rd Generation

Helicos. Pacific BiosciencesVisigGen . Nanopore(s)

www.helicosbio.com

HelicosFirst true single molecule sequencing (tSMS)

www.helicosbio.com

HelicosDRS

Nature 461:814 (2009)

www.pacificbiosciences.com

et al. : 2

Pacific BiosciencesTechnologogies

Pacific BiosciencesFluorescent lable

www.pacificbiosciences.com

Pacific BiosciencesImmobilised polymerase

long sequences

www.pacificbiosciences.com

Tex

Pacific BiosciencesReal-time DNA methylation detection

Nature Methods 7:461 (2010)

Pacific BiosciencesEpigenetics

www.pacificbiosciences.com

Pacific BiosciencesDirect RNA sequencing

www.pacificbiosciences.com

Pacific BiosciencesTranslation

www.pacificbiosciences.com

VisiGenFluorescence resonance energy transfer (FRET)

F-donor : polymeraseF-acceptor: -phosphate of dNTP

1Mb/s>1 human genome/day

visigenbio.com

NanoporeIon current

Messwert

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

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

IBMWaver pore

genome.fli-leibniz.deLecturesMaster MLS