next generation sequencing - rotes kreuz: home · semiconductor sequencing pyrosequencing...
TRANSCRIPT
![Page 1: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/1.jpg)
Next Generation Sequencing
38. Informationsgespräch der Blutspendezentrale für Wien, Niederösterreich und Burgenland
Österreichisches Rotes Kreuz
22. November 2014, Parkhotel Schönbrunn
![Page 2: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/2.jpg)
Die Zukunft hat begonnen ….
![Page 3: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/3.jpg)
Generationen
1 Sanger
2 Roche454/Illumina/IonTorrent
3 Pacific Biosystems
4 Oxford Nanopore
![Page 4: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/4.jpg)
![Page 5: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/5.jpg)
![Page 6: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/6.jpg)
![Page 7: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/7.jpg)
![Page 8: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/8.jpg)
Emulsion PCR
![Page 9: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/9.jpg)
Direct sequencing of UnseparatedAlleles
CCRCCCCGAMCGATCTACTCACTACTCACTWC
![Page 10: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/10.jpg)
Cloning and Sequencing
CCRCCCCGAMCGATCTACTCACTACTCACTWC
CCACCCCGACCGATCTACTCACTACTCACTAC
CCGCCCCGAACGATCTACTCACTACTCACTTC
![Page 11: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/11.jpg)
NGS
CCRCCCCGAMCGATCTACTCACTACTCACTWC
CCACCCC
ACCCCGACCGA
CCGAACGAT
AACGATCTACT
CTCACTACTCACTAC
TACTCACTTC
![Page 12: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/12.jpg)
Sequencing Errors
• Insertions and Deletions mainly on homopolymer stretches:
Ref: CCCCG
#1: CCCCCG
#2: CCCG
#3: CTCCG
![Page 13: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/13.jpg)
2nd Generation, Library Prep
PCR
Emulsion PCR Polony Amplification
Semiconductor Sequencing Pyro Sequencing Reversible Terminator Sequencing
![Page 14: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/14.jpg)
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+
![Page 15: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/15.jpg)
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
![Page 16: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/16.jpg)
B*57:01?
• HIV Patient from Niederösterreich
![Page 17: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/17.jpg)
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----- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
![Page 18: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/18.jpg)
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
![Page 19: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/19.jpg)
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.)
![Page 20: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/20.jpg)
Gene Conversion & Mutations
B*44:02:01:01
B*44:138Q
B*46:01:01
-281 1613 2898726
III I
![Page 21: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/21.jpg)
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
![Page 22: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/22.jpg)
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
![Page 23: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/23.jpg)
HLA Typing on a 314 Chip
![Page 24: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/24.jpg)
HLA TypeStreamT Analysis
Software
![Page 25: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/25.jpg)
Coverage
![Page 26: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/26.jpg)
• Whole gene sequencing possible
• Clonal sequences
• Automation
• Chip size
– Low to High throughput
• Costs
Advantages
![Page 27: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/27.jpg)
• HLA/IMGT database currently incomplete->?
• Urgent samples?
• Emulsion PCR?– Length of reads
– GC rich regions
– Coverage
– Phase
– Amplification bias
• -> Third Generation Sequencing?
Flaws, Outlook
![Page 28: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/28.jpg)
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+
![Page 29: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/29.jpg)
![Page 30: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/30.jpg)
![Page 31: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/31.jpg)
![Page 32: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/32.jpg)
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
![Page 33: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/33.jpg)
Schematic of the nanopore device.
Schreiber J et al. PNAS 2013;110:18910-18915
©2013 by National Academy of Sciences
![Page 34: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/34.jpg)
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
![Page 35: Next Generation Sequencing - Rotes Kreuz: Home · Semiconductor Sequencing PyroSequencing Reversible Terminator Sequencing. Sanger vsNGS (2nd Generation) Workflow ... Super high resolution](https://reader034.vdocuments.site/reader034/viewer/2022052423/5f08572b7e708231d42186b9/html5/thumbnails/35.jpg)
Die Zukunft hat begonnen ...
… und ist noch jung