14/12/2012. HLA typing - problem #1. Applications for NGS. HLA typing - problem #1 HLA typing - problem #2

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Routine HLA typing by Next Generation Sequencing

Kaimo Hirv

Center for Human Genetics and Laboratory Medicine Dr. Klein & Dr. Rost

Lochhamer Str. 29 D-82152 Martinsried Tel: 0800-GENETIK or 089-895578-0

[email protected] www.medical-genetics.de

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www.medical-genetics.de

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Applications for NGS

HLA Typing

Registry Typing

Chimerism Analysis

MRD

Cord Blood Typing

Molecular Oncology Donor Search

HLA typing - problem #1

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HLA typing - problem #1 HLA typing - problem #2

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- work-intensive

- time-consuming

- cost-intensive

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HLA typing - problem #2

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Advantages of the NGS technology - clonal amplification

less ambiguities - multiplexing

high throughput

- reduced sequencing costs per base Requirements

- high number of samples - not time critical analysis - identical requests

NGS Technologies

MiSeq Roche GS FLX Ion Torrent

Read length 150 bp 700-800 bp 200 bp

Output/run 1 GB 0,8 GB 1 GB (318)

Indexing > 100 150 > 100

Accuracy high-moderate high-moderate moderate

Cost/run 2.000 € 6.000 € 1.500 €

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Tissue Antigens 2009, 74, 393-403

Typing of

- 7 HLA Loci (A, B, C, DRB1, DQB1, DQA1, DPB1) - up to 48 DNA samples in one sequencing run

Next Gen Sequencing

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Next Gen Sequencing

Applications for NGS

HLA Typing

Registry Typing

Chimerism Analysis

MRD

Cord Blood Typing

Molecular Oncology Donor Search

Registry Typing

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GS FLX Titanium Chemistry Process Steps - Overview

DNA Library Preparation

Prepare double-stranded DNA library with adapters

Emulsion Titrations**

* One library provides enough DNA for thousands of sequencing runs.

** Only one titration is required for each sample.

Data output

Sequencing

Prepare sequencing run

Quality filtered bases 2 h prep 9 h run 3. Sequencing

emPCR

dstDNA with adaptors attached to bead

Clonally amplified sstDNA in emulsion

sstDNA ready to sequence 6 h hands on 6 h amplification 2. emPCR 2.5 h

1. Rapid Library Construction * gDNA

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Library Preparation

TS-Primer = Target-Specific Primer

ACACTGACGACATGGTTCTACACSGCCTCTGYGGGGAGAAGCA Tag Seq TS-Primer

MID-Primer = Barcode Primer

Forward

CGTATCGCCTCCCTCGCGCCATCAGACGAGTGCGTACACTGACGACATGGTTCTACA

Adaptor-Sequence Key Seq MID Seq Tag Seq

ACACTGACGACATGGTTCTACACSGCCTCTGYGGGGAGAAGCA...

TGTGACTGCTGTACCAAGATGTGSCGGAGACYCCCCTCTTCGT...

CGTATCGCCTCCCTCGCGCCATCAGACGAGTGCGTACACTGACGACATGGTTCTACACSGCCTCTGYGGGGAGAAGCA...

Pos. 4, PCR 96 Forward Primer 10µl

B1-8 in 8 Spalten

Pos. 5, PCR 96 Reverse Primer 10µl

C1-8 in 8 Spalten

Pos. 1, Matrix Mastermix 380µl

8 Tubes in Spalte 1 Pos. 2, Matrix Forward MID 20µl

96 Tubes

Pos. 3, Matrix Reverse MID 20µl

96 Tubes

Pos. 4, Matrix DNA 50µl 96 Tubes

Pos. 4 & 5 leere PCR 384 Platten

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PCR Setup

- workstation with 96 Probe Head - 384-well Thermocycler

95 DNA samples + 1 neg-control

PCR-Plates 2 x 384

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Pooling

PCR-Platte 2 x 384

1 2 3 4 5 6 7 8

- second workstation with 8 channels

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PCR Purification

1 2 3 4 5 6 7 8 1

2 3 4 5 6 7 8

- third workstation with 8 channels - Agencourt AmPure XP system

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PCR Purification

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Process Steps 2. Emulsion PCR

Clonally-amplified sstDNA attached to bead sstDNA library

Mix denatured dstDNA library with DNA Capture beads

Emulsify DNA Capture beads and PCR reagents in water- in-oil microreactors

Break microreactors and enrich for DNA- positive beads Clonal amplification occurs inside microreactors 6 h hands on 6 h amp

2 h prep 9 h run 3. Sequencing 2. emPCR

5.5 h

1. DNA Library Construction *

gDNA Data output

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Bead Enrichment

- second workstation with integrated REMe system - enrichment rate ~ 5-20%

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Process Steps

3a. Bead Deposition into PicoTiterPlate ™

Well diameter average for PicoTiterPlate is 29 µm

A single clonally amplified sstDNA bead (20 µm diameter) is deposited per well.

Layers of packing, enzyme and PPiase Beads are deposited

Plate is loaded into instrument for sequencing PTP ready for sequencing Amplified sstDNA library beads

6 h hands on 6 h amp

2 h prep 9 h run 3. Sequencing 2. emPCR

5.5 h

1. DNA Library Construction *

gDNA Data output

Each region can be loaded separately

Several gaskets can be used (2, 4, 8 and 16 regions) for the same PTP type

GS FLX Titanium Bead Deposition Loading Beads into PicoTiterPlate

Next Gen Sequencing

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Ambiguities

- Results, n=475

Unambiguous Results with „G“ Ambiguous

HLA-A 0 0,0% 475 100% 0 0%

HLA-B 2 0,4% 467 98,3% 6 1,3%

HLA-DRB1 77 16,2% 398 83,8% 0 0%

Unambiguous: e. g. DRB1*04:01:01

Ambiguities outside of exon 2 and 3: B*44:02:01G Ambiguities outside of exon 2: DRB1*11:01:01G cis-trans Ambiguities inside of exon 2 and 3:

B*15:01:01G, *35:01:01G / B*15:20, *35:43:01G

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Exon 2 Exon 3

B*15:01

Exon 2 Exon 3

B*35:01

Exon 2 Exon 3

B*35:43

Exon 2 Exon 3

B*15:20

B*07:02, *41:02 / B*40:32, *42:01 B*15:01, *40:01 / B*15:212, *40:21

Ambiguities

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Ambiguities

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Next Gen Sequencing

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Summary - HLA Typing

- HLA typing by Next Generation Sequencing is feasible in a routine usage

- ≥380 samples can be typed for HLA-A, -B and -DRB1 / run - high resolution can be achieved

- most of the results can be reported with suffix „G“

- by high number of samples, the costs are at least comparable with Sanger sequencing

- lab automation is needed for high throughput (high costs) - standards must be defined by HLA community

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Next Gen Sequencing

Ehrlich et al. BMC Genomics 2011

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Next Gen Sequencing

Ehrlich et al. BMC Genomics 2011

Center for Human Genetics and Laboratory Medicine, Dr. Klein & Dr. Rost

Lochhamer Street 29 D-82152 Martinsried

Germany

Tel: +49-800-GENETIK or +49-89-895578-0 [email protected] www.medical-genetics.de

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