DNA-Sequencing. Technologies & Devices. Matthias Platzer. Genome Analysis Leibniz Institute on Aging - Fritz Lipmann Institute (FLI)
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1 DNA-Sequencing Technologies & Devices Matthias Platzer Genome Analysis Leibniz Institute on Aging - Fritz Lipmann Institute (FLI)
2 Genome analysis DNA sequencing platforms ABI 3730xl 4/2004 & 6/ Mb/day, 850 nt reads 2 Mb/day, 550 nt reads Illumina MiSeq 1/2013 7,5 Gb/1d, 2x250 nt reads HiSeq /2013 2x300 Gb/10d, 2x100 nt reads 2x100 Gb/2d, 2x150 nt reads PacBio RS II 2/ Gb/6h, kb nt reads
3 Moore s law Next Generation Sequencing - a game changer
4 Next Generation Sequencing - NGS Terminologies 2 nd generation = massively parallel 3 rd generation = single-molecule
5 1 st Generation Maxam-Gilbert. Sanger. Pyro
6 Maxam-Gilbert sequencing Chemical modifications, radioactive *1977
7 Sanger sequencing Dideoxy chain termination, radioactive *1977 Fred Sanger Nobel prices 1958, 1980
8 Sanger sequencing Fluorescence labeling
9 Sanger sequencing Dye primer
10 Sanger sequencing Dye terminator
11 Sanger sequencing Engineered polymerase Thermo-Sequenase = Taq polymerase Phe>Thy mutated PNAS 92:6339 (1995)
12 Sanger sequencing Cycle sequencing
13 Pyrosequencing PPi ATP
14 Pyrosequencing PPi ATP
15 2 nd Generation Roche/454. Illumina/Solexa. ABI/Solid ABI/IonTorrent. Complete Genomics
16 454 Genome Sequencer GS20/FLX/FLX+ A Revolutionary Method for Rapid Whole Genome Sequencing and Assembly And more..
17 454 Nature, July
18 454 Nature, July Mb 99% accuracy 4h de novo assembly Mycoplasma genitalium (0.58 Mb): 96% coverage, 99,96% accuracy 100x currant Sanger technology
19 Roche/454 Workflow
20 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
21 Roche/454 empcr
22 Roche/454 Sample loading
23 Roche/454 Flowgram
24 Roche/454 No cloning bias green reads red - Sanger reads black - GC content G+C % GC poor low-cloned coverage GC normal high-cloned coverage
25 Roche/454 Read length
26 Advanced genetic analysis one billion bases at a time
27 Solexa Technology reversible fluorescent labels reversible 3 OH blocking
28 Illumina/Solexa Solid-phase clonal single molecule PCR Bridge amplification
29 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)
30 Illumina/Solexa Sequencing by synthesis (SBS) 1 st cycle 2 nd cycle * * *removal of fluorescent labels & 3 OH blocking
31 Illumina/Solexa Assembly - Mapping read length: nt
32 Illumina/Solexa Paired ends & Mate pairs
33 Illumina/Solexa Multiplexing
34 Illumina/Solexa Data output
35 Illumina/Solexa Patterned flow cells Patent US A1 (2012)
36 Illumina/Solexa Patterned flow cells New platforms November 2014 NextSeq Gb/day 1 human genome or 16 exomes HiSeq3000/4000 HiSeqX Ten 150 Gb/day 5 human genome at $1000 each
37 Agencourt
38 ABI/SOLID Bead deposition
39 SOLID Probes 16
40 SOLID 2-base encoding 16 dinucleotides / 4 dyes = 4 dinucleotides/dye
41 SOLID 4-color ligation
42 SOLID 4-color ligation
43 SOLID Detection
44 SOLID Cleavage
45 SOLID 2 nd cycle ligation
46 SOLID 2 nd cycle detection
47 SOLID 2 nd cycle cleavage + additional 3 ligation cyles
48 SOLID Reset after 5 th cycle
49 SOLID 6 th cycle ligation
50 SOLID Cycling scheme
51 SOLID 2-base encoding
52
53 Complete Genomics In-house technology self-assembling DNA nanoballs (DNB) patterned nanoarrays combinatorial probe anchor ligation (cpal) Drmanac et al. Science 327:78 (2009)
54 Complete Genomics - BGI Revolocity 10, genomes/year
55 PGM - Personal Genome Machine
56 Ion Torrent The chip is the machine ionogram
57 Ion Torrent Read length
58 ABI/Ion Torrent Data output
59 Ion Torrent Data output 30 Mb 1 Gb 10 Gb
60 ABI/Ion Torrent Post-light sequencing of Gordon Moore Nature 475:348 (2011)
61 2 nd Generation sequencing Applications Re-sequencing RNA-seq mirna-seq ChIP-seq Ribosome-footprinting
62 3 rd Generation Pacific Biosciences Oxford Nanopore
63
64 Pacific Biosciences Fluorescent lable
65 Pacific Biosciences Immobilised polymerase long sequences
66 Pacific Biosciences Zero-mode waveguide chip
67 Pacific Biosciences Read length P5-C3
68 Pacific Biosciences Genome de novo assemblies
69 Pacific Biosciences Read coverage
70 Pacific Biosciences Full length transcripts
71 Pacific Biosciences Alternative neurexin 1α transcripts
72 Pacific Biosciences Epigenetics
73 Pacific Biosciences Direct RNA sequencing
74 Pacific Biosciences Translation
75 nanoporetech.com
76 Oxford Nanopore Principle motor protein pore protein adapter nanoporetech.com
77 Oxford Nanopore Library nanoporetech.com
78 Oxford Nanopore Sequencing error rate: 10-30% Jain et al. Nat Methods 12:351 (2015)
79 Oxford Nanopore Data output ~ 400 Mb nanoporetech.com
80 Oxford Nanopore Data output nanoporetech.com
81 IBM Waver pore
82 genome.leibniz-fli.de Lectures
DNA-Sequencing. Technologies & Devices. Matthias Platzer. Genome Analysis Leibniz Institute on Aging - Fritz Lipmann Institute (FLI)
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2nd (Next) Generation Sequencing 2/2/2018
2nd (Next) Generation Sequencing 2/2/2018 Why do we want to sequence a genome? - To see the sequence (assembly) To validate an experiment (insert or knockout) To compare to another genome and find variations