1BLOOD CENTER FOR UPPER AUSTRIA

Eine kleine Fingerübung:RHD Genotypisierung mit

dem GS FLX(next generation sequencing)

Dr. Christian Gabriel und Stephanie Stabentheiner

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

Next generation sequencing

Total genome sequencingWhole genome sequencingMassive parallel sequencing

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

SHOT GUN SEQUENCING

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

Palacios G et al. N Engl J Med 2008;358:991-998

High-Throughput Sequencing MethodClick to b

uy N

OW!

PDF-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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)

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

AMPLICON SEQUENCING

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

TARGET BASED SEQUENCING

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

The selector and sequencing assay.

Dahl F et al. PNAS 2007;104:9387-9392

©2007 by National Academy of Sciences

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

Target based sequencing

Regionen müssen bekannt seinDNA/RNA wird aus dem Hintergrund heraushybridisiertGeeignet für:- Rekonstruktion von Haplotypen- Hochauflösende Sequenzierung aus

heterogener Zellpopulation- Transkriptome

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

12BLOOD CENTER FOR UPPER AUSTRIA

ALT: RHD Screening Strategiein Linz

Multistep Screening3 MethodenKombination derMethoden

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

1. Fragmentierungder DNA

2. Verbindung mitAdaptoren

3. Verbindung vonEinzelsträngenmit Beads überAdaptoren

4. Emulsion PCR5. Pyrosequen-

zierung6. Auswertung

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

Aufsicht auf die PicoTiterPlateTM70 x 75 mm Platte

60 x 60 mm “Chip”

1,6 Mio Wells

480 Wells/mm²

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

technicalParticularities:

Ø ~30 µm

h ~ 55 µm

vol ~ 75 pl

Querschnitt der befülltenPicoTiterPlateTM

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

EFI 2009 (O34) next generation sequence-based HLA typing Slide 6

GS-FLX Pyro-Sequencing & Base CallClick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

Messung und Datenmanagement4096x4096 Pixel CCD

~ 9 (15µm) Pixel/well32 Mbytes/Bild

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

Flowgram

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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 +

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

123

456

78

12

3

45

678

123

4

56

78

123

4

56

78

1

23

456

78

12

3

456

78

123

4

56

78

123

4

56

78

123

4

56

78

1

23

45

6

78

1

ex 1 2 3 4 5 6 7 8 9 10

MID 3MID 1

MID 2 MID 4

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

B*1801,5601

vs.B*1801,5502

163.2=Y

Sequenzierung (sequencing-based typing,SBT)

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

A+B=D+EBeispiel: HLA-BB*0702,4402B*0720, 4416B*0724, 4421

Ambiguities

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

33BLOOD CENTER FOR UPPER AUSTRIA

Phasing Probleme

RHDPsi + Weak D type 4reference

Die gibt es nicht mehr mit NGS

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

34BLOOD CENTER FOR UPPER AUSTRIA

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

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

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/

38BLOOD CENTER FOR UPPER AUSTRIA

Acknowledgements

GenomicsMartin DanzerSabine AtzmüllerChrista HacklKatja HoferNorbert NiklasHelene PolinJohannes PröllStephanie Stabentheiner

Click

to bu

y NOW

!PD

F-XChange

www.docu-track

.com C

lick t

o buy

NOW

!PD

F-XChange

www.docu-track

.com

http://www.docu-track.com/buy/http://www.docu-track.com/buy/