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/23h, 800 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

3 Moore s law Next Generation Sequencing - a game changer

4 NGS Terminologies 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 Engineered polymerase Thermo-Sequenase = Taq polymerase Phe>Thy mutated PNAS 92:6339 (1995)

12 Sanger sequencing Cycle sequencing

13 Pyrosequencing PPi ATP

14 2 nd Generation Roche/454. Illumina/Solexa. ABI/Solid ABI/IonTorrent. Complete Genomics

15 454 Genome Sequencer GS20/FLX/FLX+ A Revolutionary Method for Rapid Whole Genome Sequencing and Assembly And more..

16 454 Nature, July

17 454 Nature, July Mb 99% accuracy 4h de novo assembly Mycoplasma genitalium (0.58 Mb): 96% coverage, 99,96% accuracy 100x currant Sanger technology

18 Roche/454 Workflow

19 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

20 Roche/454 empcr

21 Roche/454 Sample loading

22 Roche/454 Flowgram

23 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

24 Roche/454 Read length

25 Advanced genetic analysis one billion bases at a time

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

27 Illumina/Solexa Linker ligation

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 Two new platforms January 2014 NextSeq Gb/day 1 human genome or 16 exomes HiSeq X Ten 150 Gb/day 5 human genome at $1000 each Patent US A1 (2012)

36 Agencourt

37 ABI/SOLID Bead deposition

38 ABI/SOLID Probes 16

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

40 ABI/SOLID 4-color ligation

41 ABI/SOLID 4-color ligation

42 ABI/SOLID Detection

43 ABI/SOLID Cleavage

44 ABI/SOLID 2 nd cycle ligation

45 ABI/SOLID 2 nd cycle detection

46 ABI/SOLID 2 nd cycle cleavage + additional 3 ligation cyles

47 ABI/SOLID Reset after 5 th cycle

48 ABI/SOLID 6 th cycle ligation

49 ABI/SOLID Cycling scheme

50 ABI/SOLID 2-base encoding

51 ABI/SOLID Paired ends two sequences

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 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

55 PGM - Personal Genome Machine

56 ABI/Ion Torrent The chip is the machine ionogram

57 ABI/Ion Torrent Read length

58 ABI/Ion Torrent Data output

59 ABI/Ion Torrent Data output

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 Helicos. Pacific Biosciences VisigGen. Nanopore(s)

63

64 Helicos First true single molecule sequencing (tsms)

65 Helicos DRS Nature 461:814 (2009)

66

67 Pacific Biosciences Technologogies et al. : 2

68 Pacific Biosciences Fluorescent lable

69 Pacific Biosciences Zero-mode waveguide chip

70 Pacific Biosciences Immobilised polymerase long sequences

71 Pacific Biosciences Read length P5-C3

72 Pacific Biosciences Read length

73 Pacific Biosciences Genome de novo assemblies

74 Pacific Biosciences Read coverage

75 Pacific Biosciences Full length transcripts

76 Pacific Biosciences Alternative neurexin 1a transcripts

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

78 Pacific Biosciences Epigenetics

79 Pacific Biosciences Direct RNA sequencing

80 Pacific Biosciences Translation

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

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

83 IBM Waver pore

84 genome.fli-leibniz.de Lectures