Disclosures for Graham Kiddle

1. Disclosures forGraham Kiddle In compliance with COI policy, ISTH requires the following disclosures to the session audience: Presentation includes discussion of the following off-label use of a drug or medical device: <N/A>

2. Thrombogenomics – raison d'être Expert curation of Genes and Causative mutations Sustainable database of reference sequences Chr17 - ITGA2B – 30 exons – Glanzmann Thrombasthenia Reducing the time to diagnosis for rare platelet and bleeding disorders Bead capture 100-200 million clusters Next Generation Sequencing Technologies Sequence Capture Technology

3. Thomas Kunicki, Willem Ouwehand, Anne Goodeve, Pieter Reitsma

4. Thrombogenomics – Curation Expert curation of Genes and Causative mutations Chr17 - ITGA2B – 30 exons – Glanzmann Thrombasthenia

5. Thrombogenomics: Curation of reference DNA sequences New Genes

6. Thrombogenomics: Website www.thrombogenomics.org.uk

7. Thrombogenomics: Curators login

8. Gene List – 78 disorders, 94 genes Acknowledgements: ISTH experts

9. Gene List – 78 disorders, 94 genes Bernard-Soulier Syndrome, DNA, transcript, 36 variants GlanzmannThrombasthenia, DNA, transcript, 223 variants Wiskott – Aldrich Syndrome, DNA, transcript, 301 variants Familial Platelet Disorder, DNA, transcript, 34 variants Acknowledgements: David Wilcox, Deborah French, Jose Lopez, Dan Bellissimo, Kathleen Freson

10. Thrombogenomics – Locus Reference Genomic Sustainable database of reference sequences

11. Locus Reference Genomic (LRG): Standard for reporting clinically relevant sequence variation in a stable coordinate system http://www.lrg-sequence.org/ European Molecular Biology Laboratory – European Bioinformatics Institute National Centre for Biotechnology Information

12. LRG: Overview • Fixed Annotation Layer • Sequence and coordinates for: • DNA • Transcripts • Translated protein • Start and stop codons • Curated clinically relevant variants • Updateable Annotation Layer • Information for mapping LRG sequence to • Genome assemblies • Legacy coordinate systems • LRG Provides • Unique, stable sequence record, ID and no versioning • Coordinate system for permanent and unambiguous reporting of clinically relevant variants • Independent of re-versioning of the genome build • Maintained in perpetuity by EBI and NCBI

13. Blood Coagulation Factors Genetic Mutation Database Project European Association for Haemophilia and Allied Disorders • Curators’ Concerns • Resource for curation • Existing databases on different software/servers • Open access • Data protection • Patient consent issues • AIMS of EAHAD Project • Bring together multiple European Databases • Common system for curation and software platform • Single portal and common format for access • Sustain and develop databases • Collaborate with ISTH and other organisations Contact for project Christopher.Ludlam@ed.ac.uk

14. Thrombogenomics – Next Generation Sequencing 100-200 million clusters Next Generation Sequencing Technologies

15. The evolution of Next Generation Sequencing (NGS) £ 2001 2013

16. UK10K Genome sequences – ITGA2B variants in 2432 genomes Variant curated as causative of GlanzmannThrombasthenia G-C Non-Synonymous SNV Synonymous E W E L N P P N I H L V P A K S K V N P L K R L F M P T 0 500 1039 Amino acid residue position

17. Next Generation Sequencing: Current platform at EASIH Illumina HiSeq 2000 Illumina GAII Illumina MiSeq Ion Torrent 318 chip The Eastern Sequence and Informatics Hub 1Gb = 109 bases

18. NGS Current platforms: Illumina A. Solid phase amplification B. 3’-blocked reversible terminators C. Reversible terminator process Incorporate all four nucleotides, each with a different dye. Wash , four-colour imaging. Cleave dye and terminating groups, wash. Repeat cycles Bentley DR, et al.,Nature, 2008;456:53-59.

19. NGS Current platforms: Ion Torrent Rothberg JM, et al.,Nature, 2011;475:348-352

20. NGS Future perspective: Oxford nanopore sequencing minION DNA sequencing instrument • No degradation in accuracy with read length • Reads both strands of intact DNA • Commercialisation planned in 2012 Clarke J, et al.,Nature nanotechnology, 2009;4:265-270

21. Thrombogenomics – Developing a diagnostic platform Bead capture Sequence Capture Technology

22. Next Generation Approach to Genetic Analysis Nicola Foad, Yagnesh Umrania

23. Capture Design • 94 Genes • All CCDS and 5' and 3' UTRs in Ensembl and RefSeq • All mutations in HGMD • TSS +/- 1000bp

24. Sequence capture workflow

25. Sequence Capture: Variable coverage High Coverage Low Coverage 5’UTR Causative SNP of thrombocytopenia and Absent Radius

26. Timescale Agree the list of genes for inclusion Jun 2012 Design and order platform Jul 2012 Enrolment of DNA samples with known mutations Curation and approval of 94 genes Delivery of platform Aug 2012 Test platform with known samples and re-balance Nov 2012 Sequence capture of 5-10 cases for each disorder Jan 2013 Sequencing and Data Analysis May 2013

27. Thrombogenomics – Summary Reference DNA sequence with respect to which a mutation is reported is crucial Database needs to be sustainable and stable for permanent and unambiguous reporting Expert curation of Genes and Causative mutations Sustainable database of reference sequences Chr17 - ITGA2B – 30 exons – Glanzmann Thrombasthenia Reducing the time to diagnosis for rare platelet and bleeding disorders Next Generation Sequencing 5 human genomes in 1 run and several orders of magnitude drop in price. Selective capture of targeted regions for many disorders and pooling of many DNA samples in one sequencing run – makes practical a multi-disorder genetic test. Bead capture 100-200 million clusters Next Generation Sequencing Technologies Sequence Capture Technology