DNA-Sequenzierung. Technologien & Geräte
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1 DNA-Sequenzierung Technologien & Geräte
2 Genome analysis DNA sequencing platforms ABI 3730xl 4/2004 & 6/ Mb/day, 850 nt reads 2 Mb/day, 550 nt reads Roche/454 GS FLX 12/ Mb/7h, 350 nt reads 800 Gb/23h, 800 nt reads Illumina/Solexa GAIIx 12/2008 & 11/ Gb/14d, 2x150 nt reads HiSeq /2010 2x150 Gb/10d, 2x100 nt reads 2x300 Gb/10d, 2x100 nt reads
3 Moore s law Next Generation Sequencing - a game changer
4 NGS Terminology 2 nd generation = massively parallel 3 rd generation = true single-molecule
5 1 st Generation Maxam-Gilbert. Sanger. Pyro
6 Maxam-Gilbert sequencing Chemical modifications, radioactive *1977
7 Maxam-Gilbert sequencing Reactions Maniatis, 1989
8 Sanger sequencing Dideoxy chain termination, radioactive *1977 Fred Sanger Nobel prices 1958, 1980
9 Sanger sequencing Fluorescence labeling
10 Sanger sequencing Dye primer
11 Sanger sequencing Dye terminator
12 Sanger sequencing Dye primer vs. terminator DP DT
13 Sanger sequencing Engineered polymerase Thermo-Sequenase = Taq polymerase Phe>Thy mutated Tabor & Richardson, PNAS 92:6339 (1995)
14 Sanger sequencing Cycle sequencing
15 Pyrosequencing PPi ATP
16 Pyrosequencing datp S is used in the pyrosequencing reaction Luciferase does not use datp S as substrate O O = P O O O = P O S O P O O O A datp S OH H
17 Pyro-Sequenzierung Demo
18 2 nd Generation 454/Roche. Solexa/Illumina. Solid Ion Torrent. Complete Genomics
19 454 Genome Sequencer GS20/FLX/FLX+ A Revolutionary Method for Rapid Whole Genome Sequencing and Assembly And more..
20 454 Nature, July
21 454 Nature, July Mb 99% accuracy 4h de novo assembly Mycoplasma genitalium (0.58 Mb): 96% coverage, 99,96% accuracy 100x currant Sanger technology
22 454 Workflow
23 454 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
24 454 empcr
25 454 Sample loading
26 454 Flowgram
27 454 No cloning bias green reads red - Sanger reads black - GC content G+C % GC poor low-cloned coverage GC normal high-cloned coverage
28 454 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
29 Roche/454 Read length
30 Roche/454 Read length
31 Advanced genetic analysis one billion bases at a time
32 Solexa Technology Clonal Singe Molecule Array TM Sequencing By Synthesis (SBS) reversible fluorescent labels reversible 3 OH blocking
33 Solexa Linker ligation
34 Solexa Solid-phase clonal single molecule PCR Bridge amplification
35 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)
36 Solexa Sequencing by synthesis (SBS) * removal of fluorescent labels & 3 OH blocking
37 Solexa Sequencing by synthesis (SBS) μm colony of 1000 singlestranded DNA templates
38 Solexa Assembly - Mapping read length: nt
39 Illumina/Solexa Paired ends & Mate pairs
40 Illumina/Solexa Multiplexing
41 Illumina/Solexa Applications Re-sequencing RNA-seq DigiTag-seq mirna-seq ChIP-seq Ribosome-footprinting
42 Illumina/Solexa Data output
43 ABI Agencourt SOLID
44 SOLID Bead deposition
45 SOLID Probes
46 SOLID 2-base encoding 16 dinucleotides / 4 dyes = 4 dinucleotides/dye
47 SOLID 4-color ligation
48 SOLID 4-color ligation
49 SOLID Detection
50 SOLID Cleavage
51 SOLID 2 nd cycle ligation
52 SOLID 2 nd cycle detection
53 SOLID 2 nd cycle cleavage + additional 3 ligation cyles
54 SOLID Reset after 5 th cycle
55 SOLID 6 th cycle ligation
56 SOLID Cycling scheme
57 SOLID 2-base encoding
58 SOLID Paired ends two sequences
59 SOLID Performance
60
61 Complete Genomics In-house technology self-assembling DNA nanoballs (DNB) patterned nanoarrays combinatorial probe anchor ligation (cpal) Drmanac et al. Science 327:78 (2009)
62 Complete Genomics Performance launched by mid : 2011: ~600 human genomes ~4,000 genomes end 2011: genomes/month mid 2012: new machines with 6 genomes/day end 2012: <3,000 $/genome
63 PGM - Personal Genome Machine
64 Ion Torrent The chip is the machine ionogram
65 Ion Torrent Read length
66 Ion Torrent Data output
67 Ion Torrent Post-light sequencing of Gordon Moore Nature 475:348 (2011)
68 3 rd Generation Helicos. Pacific Biosciences VisigGen. Nanopore(s)
69
70 Helicos First true single molecule sequencing (tsms)
71 Helicos Performance
72 Helicos DRS Nature 461:814 (2009)
73
74 Pacific Biosciences Technologogies
75 Pacific Biosciences Fluoreszensmarkierung
76 Pacific Biosciences Fluoreszensmarkierung
77 Pacific Biosciences Hintergrund-Rauschen
78 Pacific Biosciences Kleinstes Detektionsvolumen
79 Pacific Biosciences Immobilisierte Polymerase
80 Pacific Biosciences Immobilisierte Polymerase lange Sequenzen
81 Pacific Biosciences Template
82 Pacific Biosciences Error rate Tex
83 Pacific Biosciences Read length
84 Pacific Biosciences Real-time DNA methylation detection Tex Nature Methods 7:461 (2010)
85 Pacific Biosciences Epigenetics
86 Pacific Biosciences Direct RNA sequencing
87 Pacific Biosciences Translation
88 VisiGen Fluoreszens-Resonanz-Energie-Transfer (FRET) F-Donor : F-Akzeptor: Polymerase -Phosphat des dntp 1Mb/s >1 humanes Genom/Tag visigenbio.com
89 Nanopore Ion current Messwert Strom von K + -Ionen, die der DNA beim Porendurchtritt entgegen fließen
90 Nanopore Protein pore & exonuclease Clarke et al., Nature Nanotechnology 4:265(2009)
91 IBM Waver pore
92 genome.fli-leibniz.de Teaching Lectures
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