DNA-Sequencing Technologies & Devices
Genome analysis DNA sequencing platforms ABI 3730xl 4/2004 & 6/2006 1 Mb/day, 850 nt reads 2 Mb/day, 550 nt reads Roche/454 GS FLX 12/2006 800 Mb/23h, 800 nt reads Illumina MiSeq 1/2013 7,5 Gb/1d, 2x250 nt reads HiSeq 2500 1/2013 2x300 Gb/10d, 2x100 nt reads 2x100 Gb/2d, 2x150 nt reads
Moore s law Next Generation Sequencing - a game changer http://ivory.idyll.org/permanent/lstein-ngs-capacity.png
NGS Terminologies 2 nd generation = massively parallel 3 rd generation = true single-molecule
1 st 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
Sanger sequencing Engineered polymerase Thermo-Sequenase = Taq polymerase Phe>Thy mutated PNAS 92:6339 (1995)
Sanger sequencing Cycle sequencing
Pyrosequencing PPi ATP
2 nd Generation Roche/454. Illumina/Solexa. ABI/Solid ABI/IonTorrent. Complete Genomics
454 Genome Sequencer GS20/FLX/FLX+ A Revolutionary Method for Rapid Whole Genome Sequencing and Assembly And more..
454 Nature, July 31 2005
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
Roche/454 Workflow
Roche/454 Preparation of A/B-fragments for sequencing dsdna fragments P Bio A P Ligation B Bio Fill in Bio Bio Bio Capture on SA-Beads & Wash Alkaline Elution A B
Roche/454 empcr
Roche/454 Sample loading
Roche/454 Flowgram
Roche/454 No cloning bias green - 454 reads red - Sanger reads black - GC content G+C % GC poor low-cloned coverage GC normal high-cloned coverage
Roche/454 Deep sequencing rare mutation detection T only C to T ratio: 1/500 Mutation Freq. Mutation Freq. Primer sequence Background subtracted 0.16% Coverage Coverage Primer sequence
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
Illumina/Solexa Solid-phase clonal single molecule PCR 100μm colony of 1000 singlestranded DNA templates Single well of 454 Life Sciences PicoTiterPlate TM (to same scale)
Illumina/Solexa Sequencing by synthesis (SBS) 1 st cycle 2 nd cycle * *removal of fluorescent labels & 3 OH blocking *
Illumina/Solexa Sequencing by synthesis (SBS) 1 2 100μm colony of 1000 singlestranded DNA templates
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
Illumina/Solexa Patterned flow cells Two new platforms January 2014 NextSeq 500 30 Gb/day 1 human genome or 16 exomes HiSeq X Ten 150 Gb/day 5 human genome at $1000 each Patent US 20120316086 A1 (2012)
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 2 nd cycle ligation
ABI/SOLID 2 nd cycle detection
ABI/SOLID 2 nd cycle cleavage + additional 3 ligation cyles
ABI/SOLID Reset after 5 th cycle
ABI/SOLID 6 th cycle ligation
ABI/SOLID Cycling scheme
ABI/SOLID 2-base encoding
ABI/SOLID Paired ends two sequences
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: 2011: ~600 human genomes ~4,000 genomes end 2011: 800-1200 genomes/month mid 2012: new machines with 6 genomes/day end 2012: <3,000 $/genome
PGM - Personal Genome Machine www.iontorrent.com
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
ABI/Ion Torrent Post-light sequencing of Gordon Moore Nature 475:348 (2011)
2 nd Generation sequencing Applications Re-sequencing RNA-seq mirna-seq ChIP-seq Ribosome-footprinting
3 rd 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
Pacific Biosciences Technologogies et al. : 2
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
Pacific Biosciences Real-time DNA methylation detection Tex 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 : F-acceptor: polymerase 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