DNA-Sequencing. Technologies & Devices

Transcription

1 DNA-Sequencing Technologies & Devices

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 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 Dye primer vs. terminator DP DT

12 Sanger sequencing Engineered polymerase Thermo-Sequenase = Taq polymerase Phe>Thy mutated PNAS 92:6339 (1995)

13 Sanger sequencing Cycle sequencing

14 Pyrosequencing PPi ATP

15 2 nd Generation 454/Roche. Solexa/Illumina. Solid Ion Torrent. 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

19 454 Workflow

20 454 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

21 454 empcr

22 454 Sample loading

23 454 Flowgram

24 454 No cloning bias green reads red - Sanger reads black - GC content G+C % GC poor low-cloned coverage GC normal high-cloned coverage

25 454 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

26 Roche/454 Read length

27 Roche/454 Read length

28 Advanced genetic analysis one billion bases at a time

29 Solexa Technology Clonal Singe Molecule Array TM Sequencing By Synthesis (SBS) reversible fluorescent labels reversible 3 OH blocking

30 Solexa Linker ligation

31 Solexa Solid-phase clonal single molecule PCR Bridge amplification

32 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)

33 Solexa Sequencing by synthesis (SBS) * removal of fluorescent labels & 3 OH blocking

34 Solexa Sequencing by synthesis (SBS) μm colony of 1000 singlestranded DNA templates

35 Solexa Assembly - Mapping read length: nt

36 Illumina/Solexa Paired ends & Mate pairs

37 Illumina/Solexa Multiplexing

38 Illumina/Solexa Applications Re-sequencing RNA-seq DigiTag-seq mirna-seq ChIP-seq Ribosome-footprinting

39 Illumina/Solexa Data output

40 ABI Agencourt SOLID

41 SOLID Bead deposition

42 SOLID Probes 16

43 SOLID 2-base encoding 16 dinucleotides / 4 dyes = 4 dinucleotides/dye

44 SOLID 4-color ligation

45 SOLID 4-color ligation

46 SOLID Detection

47 SOLID Cleavage

48 SOLID 2 nd cycle ligation

49 SOLID 2 nd cycle detection

50 SOLID 2 nd cycle cleavage + additional 3 ligation cyles

51 SOLID Reset after 5 th cycle

52 SOLID 6 th cycle ligation

53 SOLID Cycling scheme

54 SOLID 2-base encoding

55 SOLID Paired ends two sequences

56 SOLID Performance

57

58 Complete Genomics In-house technology self-assembling DNA nanoballs (DNB) patterned nanoarrays combinatorial probe anchor ligation (cpal) Drmanac et al. Science 327:78 (2009)

59 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

60 PGM - Personal Genome Machine

61 Ion Torrent The chip is the machine ionogram

62 Ion Torrent Read length et al. : 2

63 Ion Torrent Data output et al. : 2

64 Ion Torrent Post-light sequencing of Gordon Moore Nature 475:348 (2011)

65 3 rd Generation Helicos. Pacific Biosciences VisigGen. Nanopore(s)

66

67 Helicos First true single molecule sequencing (tsms)

68 Helicos DRS Nature 461:814 (2009)

69

70 Pacific Biosciences Technologogies et al. : 2

71 Pacific Biosciences Fluorescent lable

72 Pacific Biosciences Immobilised polymerase long sequences

73 Pacific Biosciences Real-time DNA methylation detection Tex Nature Methods 7:461 (2010)

74 Pacific Biosciences Epigenetics

75 Pacific Biosciences Direct RNA sequencing

76 Pacific Biosciences Translation

77 VisiGen Fluorescence resonance energy transfer (FRET) F-donor : F-acceptor: polymerase -phosphate of dntp 1Mb/s >1 human genome/day visigenbio.com

78 Nanopore Ion current Messwert Strom von K + -Ionen, die der DNA beim Porendurchtritt entgegen fließen

79 IBM Waver pore

80 genome.fli-leibniz.de Lectures Master MLS