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Eine kleine Fingerübung · 2012. 11. 14. · 1 BLOOD CENTER FOR UPPER AUSTRIA Eine kleine...
Transcript of Eine kleine Fingerübung · 2012. 11. 14. · 1 BLOOD CENTER FOR UPPER AUSTRIA Eine kleine...
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1BLOOD CENTER FOR UPPER AUSTRIA
Eine kleine Fingerübung:RHD Genotypisierung mit
dem GS FLX(next generation sequencing)
Dr. Christian Gabriel und Stephanie Stabentheiner
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Next generation sequencing
Total genome sequencingWhole genome sequencingMassive parallel sequencing
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SHOT GUN SEQUENCING
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Palacios G et al. N Engl J Med 2008;358:991-998
High-Throughput Sequencing MethodClick to b
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Shot gun sequencing
Geeignet für:- ganze Genome,- Transkriptome- Metagenome
Eigentlich alles, wo man nicht weiß was mansucht und einmal wissen will wie das GenomaussiehtVoraussetzung: Aufreinigung der Zellen (zBKultivierung und Sorten)
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AMPLICON SEQUENCING
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Amplicon sequencing
Definierte Regionen müssen bekannt seinund werden mit PCR amplifiziertGeeignet für:- Klonale Sequenzierung- Ultra deep sequencing- Große Gene und Gencluster mit vielen Exons
und vielen Variationen
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TARGET BASED SEQUENCING
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The selector and sequencing assay.
Dahl F et al. PNAS 2007;104:9387-9392
©2007 by National Academy of Sciences
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Target based sequencing
Regionen müssen bekannt seinDNA/RNA wird aus dem Hintergrund heraushybridisiertGeeignet für:- Rekonstruktion von Haplotypen- Hochauflösende Sequenzierung aus
heterogener Zellpopulation- Transkriptome
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11BLOOD CENTER FOR UPPER AUSTRIA
RHD Genotypisierung
Partial und weak D Allele müssen unterschieden werden:- in der Schwangerenserologie und der- Transfusionsmedizin- um das Immunisierungsrisiko ein zu schätzen
Sequence based typing (SBT) mit Sanger-Methode ist derzeit derGold-Standard
Ziel: Einrichtung eines neuen RHD Genotypisierungssystems ohneLimitationen der konventionellen MethodenHoher DurchsatzNiedrige KostenIdentifikation von Insertionen, Deletionen und HybridenKlonale Sequenzierung mit allelischer Auflösung der cis/transAmbiguities
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12BLOOD CENTER FOR UPPER AUSTRIA
ALT: RHD Screening Strategiein Linz
Multistep Screening3 MethodenKombination derMethoden
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13BLOOD CENTER FOR UPPER AUSTRIA
Next-gen sequencing (NGS)3 Systeme:- SOLID (Applied Biosystems)- Genome Analyzer (Illumina Inc.)- Genome Sequencer FLX (Roche 454 Life Sciences)
Next-generation sequencing übertrifft Sanger SBTclonale SequenzierungDurchsatz
GS FLX seit Mai 2008 in LinzVorteile:
lange Leselängen(bis 500bp)Amplicon Sequenzierung
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1. Fragmentierungder DNA
2. Verbindung mitAdaptoren
3. Verbindung vonEinzelsträngenmit Beads überAdaptoren
4. Emulsion PCR5. Pyrosequen-
zierung6. Auswertung
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emulsion-PCREin Mikroreaktor
Mit einem Bead>
Mit einem Fragment>
Einem Clone>
Einer PCR>
107 Microreaktoren/ml
107 Template Kopien/bead
Bead Type A
Bead Type B
Sequencing Primer Type B
Sequencing Primer Type A
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Aufsicht auf die PicoTiterPlateTM70 x 75 mm Platte
60 x 60 mm “Chip”
1,6 Mio Wells
480 Wells/mm²
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technicalParticularities:
Ø ~30 µm
h ~ 55 µm
vol ~ 75 pl
Querschnitt der befülltenPicoTiterPlateTM
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EFI 2009 (O34) next generation sequence-based HLA typing Slide 6
GS-FLX Pyro-Sequencing & Base CallClick t
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Messung und Datenmanagement4096x4096 Pixel CCD
~ 9 (15µm) Pixel/well32 Mbytes/Bild
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Flowgram
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21BLOOD CENTER FOR UPPER AUSTRIA
NGS Primer Design
5‘ Adaptor A 19bp MID 10bp Target specific primer 18-26bp 3‘
5‘ Adaptor B 19bp MID 10bp Target specific primer 18-24bp 3‘
Implementation der Sanger SBT auf dem GS FLXFusionsprimer Design: Zielspezifische Primer identisch mitAmplifikationsprimer der Sanger SBTAnwendung der Multiplex identifier Sequenzen(MID)
primer fwd
primer rev
Damit können bis zu 26 Patienten parallel sequenziert werden4 MIDs20 Primer à ~ 52bp per MIDinsgesamt: 80 Primer
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22BLOOD CENTER FOR UPPER AUSTRIA
Amplicon Strategietarget amplicon length
D-specifity target amplicon length
D-specifity
RHD Exon 1 Sanger 767bp + RHD Exon 6 Sanger 274bp +
NGS 825bp + NGS 332bp +
RHD Exon 2 Sanger 1478bp + RHD Exon 7 Sanger 405bp +
NGS 480bp - NGS 463bp +
RHD Exon 3 Sanger 208bp + RHD Exon 8 Sanger 709bp +
NGS 266bp + NGS 767bp +
RHD Exon 4 Sanger 379bp + RHD Exon 9 Sanger 557bp +
NGS 507bp + NGS 615bp +
RHD Exon 5 Sanger 442bp + RHD Exon 10 Sanger 382bp +
NGS 500bp + NGS 440bp +
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23BLOOD CENTER FOR UPPER AUSTRIA
PatientenselektionWeak D Expression oder unklare D Phänotypen inStandardmethoden26 Patienten selektiert (25 Blutspender und einRundversuchsteilnehmer)Alle Patienten in Sanger SBT analysiert(Referenzmethode)Nachfolgende RHD Genotypisierung mit NGSVergleich Sanger SBT mit NGS SBT
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24BLOOD CENTER FOR UPPER AUSTRIA
Next-gen sequencing workflow
1. Amplicon-Generation (8 Patienten à 4MIDs in einer 96well Platte)
2. SPRI Aufreinigung der Amplicons3. Fluorescence basierte Quantifizierung4. Equimolares library pooling (3-4 Probem in
einer library)5. EmulsionsPCR ermöglicht clonale
Amplifikation der gebundenen DNAMoleküle in einem Vesikel
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78
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ex 1 2 3 4 5 6 7 8 9 10
MID 3MID 1
MID 2 MID 4
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25BLOOD CENTER FOR UPPER AUSTRIA
Sequenzierung in einer 8 regionPTP Platte (70x75mm)
pat1-4
MID 1MID 2MID 3MID 4
40ampl.
pat5-8
MID 1MID 2MID 3MID 4
40ampl.
pat9-11
MID 1MID 2MID 3
30ampl.
pat12-14
MID 1MID 2MID 3
30ampl.
pat15-17
MID 1MID 2MID 3
30ampl.
pat18-20
MID 1MID 2MID 3
30ampl.
pat21-23
MID 1MID 2MID 3
30ampl.
pat24-26
MID 1MID 2MID 3
30ampl. 26 patients
260 amplicons
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26BLOOD CENTER FOR UPPER AUSTRIA
Ergebnisse der Sanger SBT
1-4 SNPs pro ProbeSNPs verteilt über alle 10 Exons5/26 Proben ohne Veränderungen
14 verschiedene weak D Allele2 D Kategorien2 Partial D‘sRHDel (IVS3+1 G>A), RHD (W16X), RHDPsihomo-/hemi- and heterozygote Mutationen
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27BLOOD CENTER FOR UPPER AUSTRIA
Ergebnisse des NGS…
33 der 35 SNPs korrekt identifiziertKonkordanz 94,3%
Homo-/Heterozygosität in Konkordanz zwischen NGS und SangerSBT für 32 SNPs
Exon 2 nicht D spezifisch: Heterozygosität mit dem RHCE Gen
Stabentheiner et al. Vox Sang 2011
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28BLOOD CENTER FOR UPPER AUSTRIA
Es gab noch ein paarKleinigkeiten…
2 Polymorphismen in Exon 3 nicht gefundenExon 3 fwd sequence reads funktionierten in 18 der 26Proben nichtRedesign des Exon 3 PrimersZiel: vollständige Abdeckung des Exons in den forwardand reverse reads
Stabentheiner et al. Vox Sang 2011
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29BLOOD CENTER FOR UPPER AUSTRIA
Zunehmende Komplexitäten…2 Allele in komplexen Konstellationen:Weak D type 4 und D III type 6Weak D type 4 und RHDPsi
Probleme mit der Sanger SBTKeine Auflösung der cis/trans Verbindungen derPolymorphismen in einem AmpliconSignalüberlagerung in Elektropherogrammen ermöglichtkeine klare Zuordnung der Basen
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B*1801,5601
vs.B*1801,5502
163.2=Y
Sequenzierung (sequencing-based typing,SBT)
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Polymorphic positions
Core heterozygous sequence data
Exon 2 Exon 3Exon 1 Exon 4
Beispiel: HLA-BB*0702, 4402B*0702, 4419N
Mutationen liegen außerhalb dersequenzierten Bereiche
Ambiguities
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A+B=D+EBeispiel: HLA-BB*0702,4402B*0720, 4416B*0724, 4421
Ambiguities
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33BLOOD CENTER FOR UPPER AUSTRIA
Phasing Probleme
RHDPsi + Weak D type 4reference
Die gibt es nicht mehr mit NGS
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34BLOOD CENTER FOR UPPER AUSTRIA
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35BLOOD CENTER FOR UPPER AUSTRIA
IVS3-19 37bp Duplikation
von 1450 reads waren 36,2% mit einer 37bp DuplikationKeine 50% vs. 50% Verteilung (Heterozygosität):Präferentielle Amplifikation anzunehmen
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36BLOOD CENTER FOR UPPER AUSTRIA
High throughput Genotyping
Wir haben nicht alles ausgenutztEs wären (theoretisch lt. Hersteller) bis zu 500Proben möglich in einem LaufWir haben uns in der HLA Sequenzierung auf 48Patienten in der höchstmöglichen Auflösung (à 17Amplicons) eingerichtetInteressant für die ABO Genotypisierung
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37BLOOD CENTER FOR UPPER AUSTRIA
ZusammenfassungKlonale Sequenzierung mit NGS ermöglicht :- Auflösung der cis/trans Überlagerung von SNPs- Klare Identifikation der Duplikationen- de novo Veränderungen sind eindeutig identifizierbar
Automatisation ist noch offenMöglichkeiten für Hochdurchsatz absolut gegeben
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38BLOOD CENTER FOR UPPER AUSTRIA
Acknowledgements
GenomicsMartin DanzerSabine AtzmüllerChrista HacklKatja HoferNorbert NiklasHelene PolinJohannes PröllStephanie Stabentheiner
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