Next Generation Sequencing

Transcription

1 Next Generation Sequencing 38. Informationsgespräch der Blutspendezentralefür Wien, Niederösterreich und Burgenland Österreichisches Rotes Kreuz 22. November 2014, Parkhotel Schönbrunn

2 Die Zukunft hat begonnen.

3 Generationen 1 Sanger 2 Roche454/Illumina/IonTorrent 3 Pacific Biosystems 4 Oxford Nanopore

4

5

6

7

8 Emulsion PCR

9 DirectsequencingofUnseparated Alleles CCRCCCCGAMCGATCTACTCACTACTCACTWC

10 CloningandSequencing CCRCCCCGAMCGATCTACTCACTACTCACTWC CCACCCCGACCGATCTACTCACTACTCACTAC CCGCCCCGAACGATCTACTCACTACTCACTTC

11 NGS CCRCCCCGAMCGATCTACTCACTACTCACTWC CCACCCC ACCCCGACCGA CCGAACGAT AACGATCTACT CTCACTACTCACTAC TACTCACTTC

12 Sequencing Errors InsertionsandDeletionsmainlyon homopolymer stretches: Ref: CCCCG #1: CCCCCG #2: CCCG #3: CTCCG

13 2 nd Generation, Library Prep PCR Emulsion PCR Polony Amplification Semiconductor Sequencing Pyro Sequencing Reversible Terminator Sequencing

14 Sanger vsngs (2 nd Generation) Workflow PCR (Exons2, 3) Sequenzierreaktion Capillarelektophorese Amplifikation einzelner Moleküle Run, Erfassung der Rxn in ACCGGGAGACACAGATCTGCAAGGCCAAGGCGACAGACTG ACCGAGAGAACCTGCGGATCGCGCTCCGCTACTACAACCAG AGCGAGGCCGGTGAGTGACCCCGGCCCGGGGCGCAGGTC ACGACCGCCCATCCACGTACGCGGCGCCCGATC >B=@?;; =;<8:97396>>>>?@ >9 430,,00&,&-0&-00& '-25+6,,( ,2.4047*001+

15 Proof of Principle Bentley et al, Tissue Antigens 2009, 74:393 Gabriel et al, Hum Immunol2009, 70:960 Lindet al, Hum Immunol2010, 71:1033 Erlichet al, BMC Genomics2011, 12:42 Holcomb et al, Tissue Antigens 2011, 77:206 Wang et al, ProcNatlAcadSci2012,109:8676 Shiina et al, Tissue Antigens 2012, 80:305

16 B*57:01? HIV Patient from Niederösterreich

17 Deletionin Exon3 gdna B*44:138Q GGCCAG GGTC TCACA...TCCAG AGGATGTACG GCTGCGACGT GGGGCCGGAC GGGCGCCTCC TCCGCGGGTA TGACCAGGAC GCCTACGACG GCAAGGATTA B*44:02:01: TCA

18 Sanger-Primer Designation Sequence (5 ->3 ) Purpose B*44--18fwd GCA CCC ACC CGG ACT CAG AA Amplification & Sequencing B* rev GGG GTC ACG GTG GAC ACG G Amplification & Sequencing B* rev TCG TCC ACG TAG CCC ACG GT Sequencing B* fwd GGG TCT CAC ATC ATC CAG AGG Sequencing B* fwd GTC CTA GGG TGT CCC ATG AG Sequencing B* rev GAA GAG ATA TGA CCC CTC ATC Sequencing B* fwd CTG GAG CCC TTC AGC AGG Sequencing B* fwd TGT GAT GTG TAG GAG GAA GAG C Sequencing B* fwd TCC CAG TCC CCT CAC AGG G Sequencing B* rev CCC ACC CAC CCC CAG ACC T Sequencing

19 NGS-Primer Designation Sequence (5 ->3 ) Purpose B_F1 CCC GGT TGC AAT AGA CAG TAA CAA A NGS Amplification (Shiinaetal.) B_R1 GGG TCC AAT TTC ACA GAC AAA TGT NGS Amplification (Shiina et al.)

20 Gene Conversion& Mutations B*44:138Q III I B*44:02:01:01 B*46:01:01

21 Propositi Patient SN: Father SG: Sister SGr: Brother SA: HLA Typing A*02:01/09,*03:01 B*07:02,*44:138Q C*07:02,*07:04 A*23:01,*03:01 B*44:03,* 44:138Q C*04:01,*07:04 A*02:01/09,*23:01 B*07:02,*44:03 C*07:02,*04:01 A*02:01/09,*03:01 B*40:01,* 44:138Q C*03:04,*07:04

22 Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers, T. Shiina, et al. Tissue Antigens, 2012, 80,

23 HLA Typingon a 314 Chip

24 HLA TypeStreamT Analysis Software

25 Coverage

26 Advantages Whole gene sequencing possible Clonal sequences Automation Chip size Low to High throughput Costs

27 Flaws, Outlook HLA/IMGT database currently incomplete->? Urgent samples? Emulsion PCR? Length of reads GC rich regions Coverage Phase Amplification bias -> Third Generation Sequencing?

28 Sanger vsngs (3 rd Generation) Workflow PCR (Exons2, 3) Sequenzierreaktion Capillarelektophorese Amplifikation einzelner Moleküle Run, Erfassung der Rxn in ACCGGGAGACACAGATCTGCAAGGCCAAGGCGACAGACTG ACCGAGAGAACCTGCGGATCGCGCTCCGCTACTACAACCAG AGCGAGGCCGGTGAGTGACCCCGGCCCGGGGCGCAGGTC ACGACCGCCCATCCACGTACGCGGCGCCCGATC >B=@?;; =;<8:97396>>>>?@ >9 430,,00&,&-0&-00& '-25+6,,( ,2.4047*001+

29

30

31

32 Generation 4 -NanoporeSequencing Single molecule sequencing incorporating nanopore technology Protein pore->solid state channel(dna-transistor) USB size portable DNA sequencer Wholegenomescan15 min Very low cost

33 Schematic of the nanopore device. Schreiber J et al. PNAS 2013;110: by National Academy of Sciences

34 Strategy for reading epigenetic modifications on a target CG dinucleotide. Schreiber J et al. PNAS 2013;110: by National Academy of Sciences

35 Die Zukunft hat begonnen... und ist noch jung