Next Generation Sequencing

Transcription

1 Educational Session 28th EFI Conference, Stockholm Kaimo Hirv ZENTRUM FÜR HUMANGENETIK UND LABORATORIUMSDIAGNOSTIK (MVZ) Dr. Klein, Dr. Rost und Kollegen Lochhamer Str Martinsried Tel: 0800-GENETIK oder

2 NGS at the EFI conference 2014, oral presentation or poster general NGS topics 6 Illumina 8 IonTorrent 4 PacBio 5 23

3 FLX, Roche Miseq IonTorrent RS II Read length bp 2x300 bp 400 bp > 3000 bp Output / run 0.8 GB GB 1 GB GB Reads Speed Multiplexing yes yes yes yes

4 Key questions before you start Type of HLA analysis in your lab (registry typing / solid organs?) number of samples turn-around-time what are your goals high-throughput? ultra high resolution? research or clinical diagnostics? experiencies with Sanger Sequencing automation level in your lab which technology?

5 Technology target enrichment amplicon-based (PCR) hybridization PCR conventional PCR, up to 500bp (2 x 300 bp run) long-range-pcr additional fragmentation

6 Immunogenetics at Center for Human Genetics, Martinsried, Germany: Cord Blood HLA Typing Donor Search HLA Typing Chimerism Analyses Registry Typing HLA Typing HLA / Disease Association Immunodeficiencies / hereditary fever

7 NGS for Registry Typing, why? high number of samples similar HLA typing requests: A-B-DRB1 or A-B-C-DRB1-DQB1 acceptable turn-around-time, up to 3 months high-resolution typing leads to more donor work-ups limited financial resources

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9 Electronic order Sample LISS 96-well DNA plate Lab - ID CHEMAGIC STAR Barcode Scan DNA plate assignment DNA preparation 6 8

10 Standard-PCR and multiplexing PCR Master-Mix: Taq, dntp, Buffer Target-specific Primer Index 1 (fwd) Index 2 (rev) DNA

11 Standard-PCR and multiplexing target region target specific primer, fwd target specific primer, rev primer adaptor 1 primer adaptor 2 index 1, fwd index 2, rev adaptor 1 adaptor 2

12 Standard-PCR and multiplexing target region target specific primer, fwd target specific primer, rev primer adaptor 1 primer adaptor 2 index 1, fwd index 2, rev adaptor 1 adaptor 2

13 Standard-PCR and multiplexing i 1 i 9 i 2 i 10 i 3 i 11 i i i 8 i 20

14 PCR setup 3 x 384 PCR plate 96-well DNA plate unique indexes - HLA-A-B-C-DRB1-DQB1-96 x 12 PCRs per 96-well DNA plate x 12 PCRs per sequencing run (4608 reactions)

15 Pooling 3 x 384 PCR plate amplicon pools from 96 DNA samples

16 PCR Clean-up Next Generation Sequencing

17 Quantification and Loading Quantification Dilution 4 nm M Denaturation Loading M Masterpool

18 Sequencing

19 Sequencing

20 Sequencing

21 Sequencing

22 Sequencing Read 2 Adapter 2 Index-Read 2 DNA Fragment Read 1 Adapter 1 Index-Read 1 Flowcell

23 Bioinformatics / Sequence analysis software Raw data 1. Processing HLA-specific data analysis MiSeq PGM FLX FASTQ files HLA typing result SeqPilot / SeqNextHLA SBTEngine Omixon Conexio Assign NGS

24 Bioinformatics / Sequence analysis software Raw data 1. Processing HLA-specific data analysis MiSeq PGM FLX FASTQ files HLA typing result Lab IT solution: Sample-ID Index Data export to the LIS

25 Bioinformatics / Sequence analysis software Assessment of data quality alignment to the correct gene / exon? number of reads (coverage) number of reads with perfect match allele 1 / 2 ratio possibility of allele drop-out? background / contamination?

26 Bioinformatics / Sequence analysis software Sample 1 HLA-A A*02:01:01:01, A*23:17 HLA-A*02:01:01 HLA-A*23:17 HLA-B B*07:05:01, B*53:01:01 HLA-B*07:05:01 HLA-B*53:01:01 HLA-C C*06:02:01:01, C*15:05:02 HLA-C*06:02:01 HLA-C*15:05:02 DRB1 DRB1*03:01:01:01, DRB1*10:01:01 HLA-DRB1*03:01:01 HLA-DRB1*10:01:01 DQA1 DQA1*01:05, DQA1*01:05 HLA-DQA1*01:05 HLA-DQA1*01:05 DQB1 DQB1*05:01:01:01, DQB1*05:01:01:02 HLA-DQB1*02:01:01 HLA-DQB1*05:01:01 DPA1 DPA1*03:01, DPA1*03:01 HLA-DPA1*03:01 HLA-DPA1*03:01 DPB1 DPB1*105:01, DPB1*105:01 HLA-DPB1*105:01 HLA-DPB1*105:01

27 Bioinformatics / Sequence analysis software Sample 2 HLA-A A*03:42, A*24:50 HLA-A*03:01:01 HLA-A*24:53 HLA-B - HLA-B*18:01:01 HLA-B*40:02:01 HLA-C C*03:04:01:01, C*05:01:01:01 HLA-C*03:04:01 HLA-C*05:01:01 DRB1 DRB1*03:01:01:01, DRB1*13:02:01 HLA-DRB1*03:01:01 HLA-DRB1*13:02:01 DQA1 DQA1*01:02:01:04, DQA1*01:02:01:04 HLA-DQA1*01:02:01 HLA-DQA1*01:02:01 DQB1 DQB1*06:04:01, DQB1*06:09:01 HLA-DQB1*06:09 HLA-DQB1*06:09 DPA1 DPA1*01:03:01:04, DPA1*02:01:02 HLA-DPA1*01:03:01 HLA-DPA1*02:01:02 DPB1 DPB1*01:01:01, DPB1*178:01 HLA-DPB1*04:01:01 HLA-DPB1*65:01

28 Lange et al., BMC Genomics 2014 Sanger sequencing ambiguities are resolved by group specific sequencing primers (GSSP) = setup of 2 consecutive sequencing reactions and runs is needed

29 Ambiguities B*15:01 B*35:01 Exon 2 Exon 3 Exon 2 Exon 3 B*15:20 B*35:43 Exon 2 Exon 3 Exon 2 Exon 3 B*07:02, *41:02 / B*40:32, *42:01 B*15:01, *40:01 / B*15:212, *40:21

30 Ambiguities Allele Combination 1 Allele Combination 2 A*29:02:01G, *31:01:02G A*29:32, *31:06 A*68:01:01G, *68:02:01G A*68:27, *68:39 B*07:02:01G, *41:02:01 B*40:32, *42:01 * B*15:01:01G, *40:01:01G B*15:212, *40:21 B*15:01:01G, *35:01:01G B*15:20, *35:43:01G B*35:01:01G, *41:02:01 B*35:63, *35:87 * B*35:01:01G, *52:01:01G B*53:17:02, *78:02:01 * B*35:01:01G, *53:19 B*35:02:01G, *53:01:01 B*35:01:01G, *55:02:01G B*35:60, *56:09 * B*35:01:01G, *58:34 B*35:03:01G, *58:01:01G B*40:01:01G, *49:01:01 B*40:117, *50:01:01 * C*03:02:01G, *03:03:01G C*03:04:01G, *03:132 C*07:01:01G, *07:02:01G C*07:19, *07:27 *** C*12:02:01G, *16:01:01 C*12:49, *16:15:01 C*12:03:01G, *16:02:01G C*12:05, *16:15:01

31 Summary - requirements

32 Summary - requirements

33 Summary - requirements

34 Summary - requirements

35 Summary - requirements

36 Summary Advantages of NGS high resolution high throughput cost reduction Disadvantages long turn-around-time (Illumina) expensive investments in robotics and sequencing devices time-consuming implementation

37 Acknowledgements Barbara Bangol Hannah Rabenstein Ingrid Nieberle Hans Knabe MVZ Martinsried Ι Humangenetik Ι Laboratoriumsmedizin Ι Mikrobiologie Ι Pathologie Ι Transfusionsmedizin 05/14