Post on 06-May-2018
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