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Next Generation Sequencing

38. Informationsgespräch der Blutspendezentrale für Wien, Niederösterreich und Burgenland

Österreichisches Rotes Kreuz

22. November 2014, Parkhotel Schönbrunn

Die Zukunft hat begonnen ….

Generationen

1 Sanger

2 Roche454/Illumina/IonTorrent

3 Pacific Biosystems

4 Oxford Nanopore

Emulsion PCR

Direct sequencing of UnseparatedAlleles

CCRCCCCGAMCGATCTACTCACTACTCACTWC

Cloning and Sequencing

CCRCCCCGAMCGATCTACTCACTACTCACTWC

CCACCCCGACCGATCTACTCACTACTCACTAC

CCGCCCCGAACGATCTACTCACTACTCACTTC

NGS

CCRCCCCGAMCGATCTACTCACTACTCACTWC

CCACCCC

ACCCCGACCGA

CCGAACGAT

AACGATCTACT

CTCACTACTCACTAC

TACTCACTTC

Sequencing Errors

• Insertions and Deletions mainly on homopolymer stretches:

Ref: CCCCG

#1: CCCCCG

#2: CCCG

#3: CTCCG

2nd Generation, Library Prep

PCR

Emulsion PCR Polony Amplification

Semiconductor Sequencing Pyro Sequencing Reversible Terminator Sequencing

Sanger vs NGS (2nd Generation)

Workflow

PCR (Exons 2, 3)

Sequenzierreaktion

Capillarelektophorese

Amplifikation einzelner Moleküle

Run, Erfassung der Rxn in Echtzeit

@YSEVQ:4:21ACCGGGAGACACAGATCTGCAAGGCCAAGGCGACAGACTGACCGAGAGAACCTGCGGATCGCGCTCCGCTACTACAACCAGAGCGAGGCCGGTGAGTGACCCCGGCCCGGGGCGCAGGTCACGACCGCCCATCCACGTACGCGGCGCCCGATC+@@9>>4;;;45276649/3.307.73;592++,9:8;97<:=;AABBBB>B=@?;;4944188889399=;<8:97396>>>>?@99393929>9430,,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 Immunol 2009, 70:960

• Lind et al, Hum Immunol 2010, 71:1033

• Erlich et al, BMC Genomics 2011, 12:42

• Holcomb et al, Tissue Antigens 2011, 77:206

• Wang et al, Proc Natl Acad Sci 2012,109:8676

• Shiina et al, Tissue Antigens 2012, 80:305

B*57:01?

• HIV Patient from Niederösterreich

Deletion in Exon 3

• 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 (Shiina et al.)

B_R1 GGG TCC AAT TTC ACA GAC AAA TGT NGS Amplification (Shiina et al.)

Gene Conversion & Mutations

B*44:02:01:01

B*44:138Q

B*46:01:01

-281 1613 2898726

III I

Propositi HLA Typing

Patient SN: A*02:01/09,*03:01

B*07:02,*44:138Q

C*07:02,*07:04

Father SG: A*23:01,*03:01

B*44:03,* 44:138Q

C*04:01,*07:04

Sister SGr: A*02:01/09,*23:01

B*07:02,*44:03

C*07:02,*04:01

Brother SA: 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 l evel using next generation sequencers , T. Shiina, et al. Tissue Antigens, 2012, 80, 305–316

HLA Typing on a 314 Chip

HLA TypeStreamT Analysis

Software

Coverage

• Whole gene sequencing possible

• Clonal sequences

• Automation

• Chip size

– Low to High throughput

• Costs

Advantages

• HLA/IMGT database currently incomplete->?

• Urgent samples?

• Emulsion PCR?– Length of reads

– GC rich regions

– Coverage

– Phase

– Amplification bias

• -> Third Generation Sequencing?

Flaws, Outlook

Sanger vs NGS (3rd Generation)

Workflow

PCR (Exons 2, 3)

Sequenzierreaktion

Capillarelektophorese

Amplifikation einzelner Moleküle

Run, Erfassung der Rxn in Echtzeit

@YSEVQ:4:21ACCGGGAGACACAGATCTGCAAGGCCAAGGCGACAGACTGACCGAGAGAACCTGCGGATCGCGCTCCGCTACTACAACCAGAGCGAGGCCGGTGAGTGACCCCGGCCCGGGGCGCAGGTCACGACCGCCCATCCACGTACGCGGCGCCCGATC+@@9>>4;;;45276649/3.307.73;592++,9:8;97<:=;AABBBB>B=@?;;4944188889399=;<8:97396>>>>?@99393929>9430,,00&,&-0&-00&.86893332..-.'-25+6,,(+1011--,2.4047*001+

Generation 4 - Nanopore Sequencing

Single molecule sequencing incorporating nanopore technology

Protein pore ->solid state channel (DNA-transistor)

USB size portable DNA sequencer

Whole genome scan 15 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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