Shifting the Paradigm in Translational and Clinical Research

Transcription

1 Science Webinar Series Exome Sequencing in Today s Lab Shifting the Paradigm in Translational and Clinical Research 4 December, 2013 Change the size of any window by dragging the lower right corner. Use controls in top right corner to close or maximize each window. What each widget does: shows the video screen opens the Ask a Question box download slides and more info shows slide window shows speaker bios search Wikipedia Facebook login Twitter login (#ScienceWebinar) LinkedIn login if you need help

2 Science Webinar Series Exome Sequencing in Today s Lab Shifting the Paradigm in Translational and Clinical Research 4 December, 2013 Brought to you by the Science/AAAS Custom Publishing Office Participating Experts: Christian Marshall, Ph.D. The Hospital for Sick Children Toronto, Canada Christian Gilissen, Ph.D. Radboud University Medical Centre Nijmegen The Netherlands Sponsored by:

3 Whole Exome Sequencing in Clinical Research Christian Marshall McLaughlin Centre, University of Toronto The Centre for Applied Genomics, The Hospital for Sick Children AAAS Technology webinar December 4 th, Advancing genomic medicine through research and education 3

4 Number of samples Genome wide molecular studies in clinical research at Sickkids Hospital Year Research microarrays (DNA) Clinical microarrays Exome sequences Whole genome sequences Data from The Centre for Applied Genomics (TCAG) and Sickkids Clinical lab Several joint research projects (TCAG, Diagnostics, Clinical Genetics) exploring the utility of using whole exome and whole genome sequencing in the future diagnosis of pediatric cases

5 Genome wide sequencing is becoming less expensive than existing genetic tests 30,000 25,000 Cost per Human Genome Cost range Existing Genetic Tests Primary Ciliary Dyskinesia, $10,000 Spinocerebellar Ataxia, $9,000 $ Reagent Cost 20,000 15,000 10,000 X linked ID, $4,000 Cardiomyopathy, $4,000 5,000 Periodic Fever Syndrome, $ Cost of WES using Ion Proton AmpliSeq in our facility is $ Microarray, $900

6 Clinical Research at Sickkids Several ongoing clinical research projects aimed at providing evidence for the introduction of whole genome sequencing (WGS) into the future clinical care of children at Sickkids: 1. Panel testing (in silico panels) 2. Clinical research genomes (Autism Project) WGS is not yet feasible to do in house (cost and turnaround time) so also using whole exome sequencing (WES) WES with Ion Proton Sequencer for: An alternative to targeted gene panel testing as part of clinical research Development of Clinical research exomes for the Autism Genome Project

7 Spectrum of Research Analysis Single Gene Analysis Gene Panels Exome Genome Increasing data Increasing complexity with exomes and genomes, more data and more need for interpretation

8 Spectrum of Research Analysis Single Gene Analysis Gene Panels Exome Genome Increasing tests New genes discovered means increasing number of tests Genetic testing is available for over 2000 rare and common conditions and the list of genes is growing

9 Why whole exome sequencing? Compared to Gene Panel testing: A single test (streamline experiments) Pre designed kits and workflows Gene panel negative can reflex to related genes/phenotypes Compared to Genome Sequencing: The majority of disease causing variants in research are in exons Less data to transfer, analyze and interpret More cost effective with a faster turnaround time

10 Exome Sequencing Timeline Comparison 5 25 Days Ion Proton AmpliSeq Ion Proton TargetSeq Solid SureSelect 0 Library prep and capture Bead/cluster prep Sequencing Workflow Ref mapping and variant calling Advances in technology and library preparation and target enrichment have made WES extremely fast and cost effective

11 Ion AmpliSeq Exome Exome sequencing with the simplicity, specificity, and speed of PCR Fast, simple and specific > 1 hour hands on time Efficient and uniform > two exomes per P1V2 chip gives >90% of bases covered at 20X Automated analysis > obtain annotated, filtered variants Construct Library Prepare Template Run Sequence Analyze data 8hr 9hr 3.5 hr 12 hr ~294,000 primer pairs across 12 primer pools Total DNA input as low as 50 ng Covers >97% of CCDS >19,000 coding genes, >198,000 coding exons, no UTRs, mirnas, or ncrnas Amplicon size range bp

12 Exome Sequencing Analysis 2 samples/p1v2 Read Generation Torrent Server 3.6 Remove Poor Reads Alignment Assembly Read Mapping Variant calling Coverage Analysis Annotation Variant Effects Frequencies Ion Reporter Variant Analysis Variant Filtering Variant Confirmation Variant annotation and analysis with Ion Reporter and Custom Pipeline with interpretation based on disease transmission

13 Ion Reporter Basic Filtering View variants and annotations. Drill deeper when needed.

14 Gene Panel Research Study Retrospective Genetic Research Samples (n=25) Referral Phenotypes Number Nephrology Focal segmental glomerulosclerosis (FSGS) 1 Ophthalmology Metabolics/Geneti cs Cone rod dystrophy, Ocular albinism, Stargardt, Dystonia, Mitochondrial, cerebellar atrophy, glycosylation disorder Neurology Epilepsy 3 Cardiology Hypertrophic cardiomyopathy 1 Immunology Period Fever Syndrome All sent for WGS through Complete Genomics and also sequenced with Ion Proton at The Centre for Applied Genomics

15 Panel Sequencing with WES WGS or WES Known Gene? (in silico panel) YES Genetic analysis (validation in CLIA lab) NO Other Known? NO YES Refinement of analysis and/or expansion of phenotype Novel Disorder? Gene Discovery For WES we are using Ion Proton as a rapid and low cost approach to sequence genes quickly

16 Panel Sequencing with WES Case Examples Prior Clinical Diagnosis Focal segmental glomerulosclerosis (FSGS) Gene Panel Results WES Results Comments ve +ve; PLCE1 9 variants in panel genes, PLCE1 fits prior diagnosis and outside panel Cone rod dystrophy +ve; PROM1 +ve; PROM1 16 variants in panel genes, PROM1 variants detected + CACNA1F Adams Oliver Syndrome N/A ve + candidates ACVR1 variant causing related disorder Good concordance of calls from gene panel and proton WES In some cases WES picked up variants outside the panels that may be contributing to the clinical presentation

17 Adams Oliver Syndrome (AOS) Sample from a subject with a prior diagnosis of Adams Oliver Syndrome (AOS) Adams Oliver syndrome (AOS) is characterized by the congenital absence of skin, known as 'aplasia cutis congenita,' usually limited to the scalp vertex, and transverse limb defects WES of the proband revealed that the known genes, ARHGAP31, RBPJ, DOCK6, EGOT did not harbour mutations that explained the phenotype

18 Trio Analysis in Ion Reporter Assuming dominant new mutation quickly use IR4.0 interface to filter and find a G328E mutation in conserved exon 8 of ACVR1 was a plausible candidate ACVR1 mutations associated with "Fibrodysplasia ossificans progressiva (FOP); features overlap with AOS and after revisiting the phenotype, the clinical presentation in this subject is consistent with a variant of FOP

19 Overview of Autism Project Design Canadian Autism Genome project High resolution SNP microarray and Whole Exome sequencing (WES) and genome (WGS) workflow N= >2000 Genomic DNA of families Illumina 2.5M Affymetrix cytohd High resolution SNP microarray N=200 High Throughput Sequencing N=600 CGI Illumina Whole genome Sequencing OR Whole Exome Sequencing Solid 5500xL Ion Proton Copy number Variation Copy Number Variation SNVs and Indels Combined High Resolution Genome Analysis Genotype Phenotype Development of Clinical research exome reports for the Autism Genome Project

20 Autism Project Design and Results Newfoundland cohort pilot (Bridget Fernandez) with 75 trios run on Solids 5500xL and 75 trios with Ion Proton: Annotated SNVs and INDELS De novo variant NO ASD/Cognit ive NO YES YES <1% AF in all databases? NO Further Research, Gene Discovery, Secondary Findings YES Variant Deleterious? VUS YES Deleterious related to phenotype VUS related to the phenotype Variants of Primary interest Segregation and Genotype Phenotype correlation Using a list of ~125 ASD candidate genes and/or de novo analysis Typically finding ~25% of cases have a variant (LOF or de novo) that may be related to the disorder (eg. NRXN1, CHD7, SCN2A, NRG4, RIMS2)

21 Inherited variants play a role in Autism NRXN1 G989* Whole Exome Sequencing: NRXN1 exon 15 Gly989stop (chr2: 50,724,505 C>A) Neurexin 1 nervous system cell adhesion molecule and ASD candidate gene NRXN1 G989* INTERPRETATION: maternally inherited NRXN1 G989* may be pathogenic variant in this family

22 Interpretation depends on technology 15q11.2 loss NRXN1 G989* NRXN1 G989* 15q11.2 loss Whole Exome Sequencing: NRXN1 exon 15 Gly989stop (chr2: 50,724,505 C>A) Neurexin 1 nervous system cell adhesion molecule and ASD candidate gene Microarray CNV analysis: 15q kb loss (HERC2P2, CYFIP1, NIPA2, NIPA1, TUBGCP5, WHDC1L1, GOLGA9P) Known ASD association with variable expressivity INTERPRETATION: NRXN1 G989* may be pathogenic variant with 15q11.2 CNV also contributing to phenotype

23 Summary and Observations Sickkids has several Research Projects aimed at testing the utility of using whole genome sequencing in future diagnostics Cost of WES and WGS is becoming less expensive than current genetic tests We are using the Ion Proton Sequencer an alternative to traditional targeted gene panel sequencing for clinical research and also for clinical research exomes Results show good concordance with gene panel testing and offer ability to find other variants possibly contributing to the phenotype (use as a tool early in diagnostics) Currently testing the yield of WES in complex neurological disorders like ASD Integration of Copy number variation is important > WGS

24 Acknowledgements The Centre for Applied Genomics The Hospital for Sick Children Stephen W. Scherer Lynette Lau Sergio Pereira Bhooma Thiruv Daniele Merico Susan Walker Kristiina Tammimies Ryan Yuen Life Technologies Karen Keith, Matt Dyer, Yang Wang, Michael Lelivelt, Fiona Hyland, Michael Gallad, Kate Rhodes, Mathieu Lariviere Sickkids Clinicians Roberto Mendoza Tino Picisone Christoph Litch Elise Heon Autism Project Collaborators Peter Szatmari (McMaster) Wendy Roberts (Sickkids), John Vincent (CAMH), Bridget Fernandez (MUN), Evdokia Anagnostou (Bloorview), Lonnie Zwaigenbaum (Univ of Alberta) Funding: Genome Clinic Project Ronald Cohn Stephen Meyn Sarah Bowdin Ronald Cohn Nasim Monfarad Peter Ray James Stavropoulos Ion Torrent products are for Research Use Only. Not for use in diagnostic procedures. Ion AmpliSeq, Proton, and PGM are trademarks of Life Technologies Corporation.

25 Science Webinar Series Exome Sequencing in Today s Lab Shifting the Paradigm in Translational and Clinical Research 4 December, 2013 Brought to you by the Science/AAAS Custom Publishing Office Participating Experts: Christian Marshall, Ph.D. The Hospital for Sick Children Toronto, Canada Christian Gilissen, Ph.D. Radboud University Medical Centre Nijmegen The Netherlands Sponsored by:

26 Exome Sequencing in Today s Lab: Shifting the Paradigm in Translational and Clinical Research Christian Gilissen PhD christian.gilissen@radboudumc.nl

27 Human genetics Nijmegen Research Clinical genetics Genome diagnostics

28 Why exome sequencing? Very accurate Sanger Targeted Exome Genome Cheap per exon High turn around Optimization possible Low chance of incidental findings Easy analysis Easy interpretation No bias for genes Standardized workflow Re use of performed exomes to interpret new ones Simple to add new genes No bias in what you sequence Little technical biases Allows detection of SVs and SNVs in one experiment Low diagnostic yield for genetically heterogeneous diseases Design and re design required Different designs for different disorders Sequencing bias No non coding regions Incidental findings Data analysis bottleneck Interpretation of noncoding variants Sufficient patients required Expensive, timeconsuming

29 Approaches Gene package approach Most genes known Trio approach Most genes unknown Variants patient Gene package Variants in known genes Pilot study: 50 exomes for 5 disorders De novo variants Pilot study: 100 trios for intellectual disability Neveling et al. Hum mut De Ligt et al. NEJM, 2012

30 Workflow Enrichment Enrichment (Agilent v4) ~ 21,000 genes Secondary Analysis Quality control Sample mix up check Variant annotation Sequencing Sequencing at BGI Copenhagen Using Illumina 2x100bp, 75x median coverage Interpretation Gene package visualization Standardized interpretation protocol Independent interpretation by 2 people Primary Analysis Read mapping with BWA Variant calling with GATK Report Validation by Sanger [Segregation analysis and functional confirmation] Report of results

31 Quality control Raw sequence and mapping statistics FastQC tool Bedtools coverage statistics Per gene / exon target coverage Variant statistics: Overlap dbsnp Number of truncating mutations Tr/Ti ratio % 90.00% 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00%

32 Sample mix up 2.5 Gender check: Calculate chry/chrx target coverage ratio Coverage ratio SNP Test: 12 common SNPs tested separately by Sanger sequencing 0 F FMMMMMF FMMFMMF F FMFMF FMFMF F FMF F FMM Gender according to patient database (M=male, F = Female) Trio check: Compare high quality variant calls between child and parents no swap patient - parent swap parent - unrelated swap child - unrelated swap

33 How to do 400 samples per month? Variants and annotation Quality control Filtering Patient DB Variant DB

34 Pilot study gene package approach 250 exomes: 50 exomes for 5 genetically heterogeneous diseases Gene package design: Only known genes are allowed, no candidate disease genes Gene lists must be up to date and is updated every ~3 months Created by team of experts from clinic, diagnostic and research division Number of genes (Sept. 2011) Blindness 144 Deafness 98 Early onset colorectal cancer 115 Mitochondrial disorders 207 Movement disorders 152 blind deaf move mito Neveling et al. Hum mut. 2013

35 Yield 29% 60% 50% 33% 44% 52% % = Maximum % of cases solved if all available genes had been Sanger sequenced 40% 30% 20% 10% 5% 8% 20% 10% 25% 11% 0% 16% 0% 0% 3% Sanger Exome 0% Neveling et al. Hum mut. 2013

36 Current packages # genes in disease package

37 Current packages # genes in disease package

38 Current packages # genes in disease package Exome sequencing can be cost-efficient 265 compared to Sanger when sequencing 3 genes or more

39 Current packages # genes in disease package

40 Pilot study de novo approach 100 patients parents! Severe intellectual disability (IQ<50) No etiological or syndromic diagnosis Negative family history Patients have reached the end stage of conventional strategies Targeted gene tests negative Genomic array profile negative De Ligt et al. NEJM, 2012

41 Yield in 100 ID patients Positive diagnosis June 2012 June 2013 All mutations De novo mutations Autosomal dominant X linked 2 4 Autosomal recessive 1 1 Inherited mutations 3 1 X linked 3 1 Autosomal recessive 0 0 Candidates Yield of ~30% in patients with severe ID De Ligt et al. NEJM, 2012

42 Example power of the exome Patient phenotype (4 years old) Delayed development, mainly speech (1 2 words) Eczema from 6 months of age Behavioral problems; aggressive, self mutilation Short stature ( 2.5 SD), OFC normal ( 1.5 SD) bilateral hypoplastic nail of 5 th toe MRI brain normal 1. Initial exome sequencing analysis of trio did not identify a cause for the disorder! 2. Open the exome : Look for de novo mutations outside of the package

43 Open the exome Nat gen Matching phenotype

44 Conclusions Exome sequencing results in a higher yield for genetically heterogeneous diseases than Sanger based approaches De novo mutations are a common cause of severe ID Future directions: Packages for many more diseases Opening the exome Genetic testing much earlier in the clinical research process Proof of Concept: Whole genome sequencing for clinical research

45 Acknowledgments Acknowledgments Kornelia Neveling Lisenka Vissers Alex Hoischen Joep de Ligt Ilse Feenstra Bregje van Bon Joris Veltman Marcel Nelen Bert de Vries Han Brunner Hans Scheffer ALL PATIENTS, PARENTS & CLINICIANS WORLDWIDE! Genome Diagnostics Helger Yntema Erik Jan Kamsteeg Lies Hoefsloot Willy Nillesen Marjolijn Ligtenberg Arjen Mensenkamp Dorien Lugtenberg Rolph Pfundt Genome Research Rick de Reuver Marisol del Rosario Nienke Wieskamp Thessa Kroes Petra de Vries Michael Kwint Irene Janssen Marloes Steehouwer Clinical genetics Marjolein Willemsen Tjitske Kleefstra Ernie Bongers David Koolen Anneke Vulto van Silfthout Wendy van Zelst Stams Sascha Vermeer

46 Science Webinar Series Exome Sequencing in Today s Lab Shifting the Paradigm in Translational and Clinical Research 4 December, 2013 Brought to you by the Science/AAAS Custom Publishing Office Participating Experts: Christian Marshall, Ph.D. The Hospital for Sick Children Toronto, Canada Christian Gilissen, Ph.D. Radboud University Medical Centre Nijmegen The Netherlands To submit your questions, type them into the text box and click. Sponsored by:

47 Science Webinar Series Exome Sequencing in Today s Lab Shifting the Paradigm in Translational and Clinical Research 4 December, 2013 Brought to you by the Science/AAAS Custom Publishing Office Look out for more webinars in the series at: webinar.sciencemag.org To provide feedback on this webinar, please e mail your comments to webinar@aaas.org For information related to this webinar, go to: Sponsored by: