Post on 18-Jun-2020
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
Die Zukunft hat begonnen ...
… und ist noch jung