Next Generation Sequencing

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

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

Emulsion PCR

DirectsequencingofUnseparated Alleles CCRCCCCGAMCGATCTACTCACTACTCACTWC

CloningandSequencing CCRCCCCGAMCGATCTACTCACTACTCACTWC CCACCCCGACCGATCTACTCACTACTCACTAC CCGCCCCGAACGATCTACTCACTACTCACTTC

NGS CCRCCCCGAMCGATCTACTCACTACTCACTWC CCACCCC ACCCCGACCGA CCGAACGAT AACGATCTACT CTCACTACTCACTAC TACTCACTTC

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

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

Sanger vsngs (2 nd Generation) Workflow PCR (Exons2, 3) Sequenzierreaktion Capillarelektophorese Amplifikation einzelner Moleküle Run, Erfassung der Rxn in Echtzeit @YSEVQ:4:21 ACCGGGAGACACAGATCTGCAAGGCCAAGGCGACAGACTG ACCGAGAGAACCTGCGGATCGCGCTCCGCTACTACAACCAG AGCGAGGCCGGTGAGTGACCCCGGCCCGGGGCGCAGGTC ACGACCGCCCATCCACGTACGCGGCGCCCGATC + @@9>>4;;;45276649/3.307.73;592++,9:8;97<:=;AABBBB >B=@?;;4944188889399=;<8:97396>>>>?@99393929>9 430,,00&,&-0&-00&.86893332..-.'-25+6,,(+1011--,2.4047*001+

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

B*57:01? HIV Patient from Niederösterreich

Deletionin Exon3 gdna 720 730 740 750 760 770 780 790 800 810 B*44:138Q GGCCAG GGTC TCACA...TCCAG AGGATGTACG GCTGCGACGT GGGGCCGGAC GGGCGCCTCC TCCGCGGGTA TGACCAGGAC GCCTACGACG GCAAGGATTA B*44:02:01:01 ------ ---- -----TCA----- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Sanger-Primer Designation Sequence (5 ->3 ) Purpose B*44--18fwd GCA CCC ACC CGG ACT CAG AA Amplification & Sequencing B*44-3347rev GGG GTC ACG GTG GAC ACG G Amplification & Sequencing B*44-559 rev TCG TCC ACG TAG CCC ACG GT Sequencing B*44-1034fwd GGG TCT CAC ATC ATC CAG AGG Sequencing B*44-1830fwd GTC CTA GGG TGT CCC ATG AG Sequencing B*44-2155rev GAA GAG ATA TGA CCC CTC ATC Sequencing B*44-2182fwd CTG GAG CCC TTC AGC AGG Sequencing B*44-2346fwd TGT GAT GTG TAG GAG GAA GAG C Sequencing B*44-2797fwd TCC CAG TCC CCT CAC AGG G Sequencing B*44-3041rev CCC ACC CAC CCC CAG ACC T Sequencing

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.)

Gene Conversion& Mutations B*44:138Q -281 726 1613 2898 III I B*44:02:01:01 B*46:01:01

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

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, 305 316

HLA Typingon a 314 Chip

HLA TypeStreamT Analysis Software

Coverage

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

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

Sanger vsngs (3 rd Generation) Workflow PCR (Exons2, 3) Sequenzierreaktion Capillarelektophorese Amplifikation einzelner Moleküle Run, Erfassung der Rxn in Echtzeit @YSEVQ:4:21 ACCGGGAGACACAGATCTGCAAGGCCAAGGCGACAGACTG ACCGAGAGAACCTGCGGATCGCGCTCCGCTACTACAACCAG AGCGAGGCCGGTGAGTGACCCCGGCCCGGGGCGCAGGTC ACGACCGCCCATCCACGTACGCGGCGCCCGATC + @@9>>4;;;45276649/3.307.73;592++,9:8;97<:=;AABBBB >B=@?;;4944188889399=;<8:97396>>>>?@99393929>9 430,,00&,&-0&-00&.86893332..-.'-25+6,,(+1011--,2.4047*001+

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

Schematic of the nanopore device. Schreiber J et al. PNAS 2013;110:18910-18915 2013 by National Academy of Sciences

Strategy for reading epigenetic modifications on a target CG dinucleotide. Schreiber J et al. PNAS 2013;110:18910-18915 2013 by National Academy of Sciences

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