Shifting the Paradigm in Translational andClinical Research
Shifting the Paradigm in Translational andClinical Research
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4 December, 20134 December, 2013
Exome Sequencing in Today’s LabExome Sequencing in Today’s LabWebinar SeriesWebinar SeriesScienceScience
Sponsored by:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
Christian Gilissen, Ph.D.Radboud University Medical Centre NijmegenThe Netherlands
Christian Marshall, Ph.D.The Hospital for Sick ChildrenToronto, Canada
Shifting the Paradigm in Translational andClinical Research
Shifting the Paradigm in Translational andClinical Research 4 December, 20134 December, 2013
Exome Sequencing in Today’s LabExome Sequencing in Today’s LabWebinar SeriesWebinar SeriesScienceScience
Advancing genomic medicine through research and educationwww.tcag.ca
AAAS – Technology webinarDecember 4th, 2013
Whole Exome Sequencing in Clinical Research
McLaughlin Centre, University of Toronto The Centre for Applied Genomics, The Hospital for Sick Children
3
Christian Marshall
• 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
Genome‐wide molecular studies in clinical research at Sickkids Hospital
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
2004 2005 2006 2007 2008 2009 2010 2011 2012
Num
ber o
f sam
ples
Year
Research microarrays(DNA)
Clinical microarrays
Exome sequences
Whole‐genomesequences
0
5,000
10,000
15,000
20,000
25,000
30,000
Cost per Human Genome
2011
2010
2012
$ Re
agen
t Cost
Cost range
Existing Genetic Tests
• Cost of WES using Ion Proton™ AmpliSeq™ in our facility is $650
Genome wide sequencing is becoming less expensive than existing genetic tests
Primary Ciliary Dyskinesia, $10,000
Cardiomyopathy, $4,000
Spinocerebellar Ataxia, $9,000
Periodic Fever Syndrome, $1500
X‐linked ID, $4,000
Microarray, $900
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
GenomeSingle Gene Analysis ExomeGene Panels
Spectrum of Research Analysis
• Increasing complexity with exomes and genomes, more data and more need for interpretation
Increasing data
GenomeSingle Gene Analysis ExomeGene Panels
Spectrum of Research Analysis
• 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
Increasing tests
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
Exome Sequencing Timeline Comparison
0
1
2
3
4
5
Library prepand capture
Bead/clusterprep
Sequencing Ref mappingand variantcalling
Days
Workflow
25
• Advances in technology and library preparation and target enrichment have made WES extremely fast and cost effective
Ion Proton™ AmpliSeq™
Ion Proton™ TargetSeq™
Solid SureSelect
Exome sequencing with the simplicity, specificity, and speed of PCR
Ion AmpliSeq™ Exome
Construct Library Run Sequence Prepare Template Analyze data8 hr 9 hr 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 225‐275 bp
• 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
Exome Sequencing Analysis
• Variant annotation and analysis with Ion Reporter and Custom Pipeline with interpretation based on disease transmission
Annotation
Alignment
Variant calling
Read Generation
Variant Analysis
Remove Poor Reads
Torrent Server 3.6
AssemblyRead Mapping
Coverage Analysis
Variant EffectsFrequencies
Variant FilteringVariant Confirmation
2 samples/P1V2
Ion Reporter
View variants and annotations. Drill deeper when needed.
Ion Reporter Basic Filtering
Retrospective Genetic Research Samples (n=25)Referral Phenotypes Number
Nephrology Focal segmental glomerulosclerosis (FSGS) 1
Ophthalmology Cone‐rod dystrophy, Ocular albinism, Stargardt,
11
Metabolics/Genetics
Dystonia, Mitochondrial, cerebellar atrophy, glycosylation disorder
7
Neurology Epilepsy 3
Cardiology Hypertrophic cardiomyopathy 1
Immunology Period Fever Syndrome 2
• All sent for WGS through Complete Genomics and also sequenced with Ion Proton at The Centre for Applied Genomics
Gene Panel Research Study
Panel Sequencing with WES
Known Gene? (in silico panel)
Other Known?Refinement of analysis and/or expansion of phenotype
Novel Disorder?
Genetic analysis (validation in CLIA lab)YES
NO
NO
YES
Gene Discovery
• For WES we are using Ion Proton as a rapid and low cost approach to sequence genes quickly
WGS or WES
Prior Clinical Diagnosis
Gene Panel Results
WES Results Comments
Focal segmental glomerulosclerosis(FSGS)
‐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
Panel Sequencing with WES
Case Examples
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
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
Overview of Autism Project Design
Genomic DNA of families
High Throughput Sequencing
Whole ExomeSequencing
Copy number Variation
Combined High Resolution Genome AnalysisGenotype‐Phenotype
• Canadian Autism Genome project High resolution SNP microarray and Whole Exome sequencing (WES) and genome (WGS) workflow
High resolution SNP microarray
Illumina 2.5MAffymetrix cytoHD
Solid 5500xL Ion Proton
Whole genome Sequencing
SNVs and IndelsCopy Number Variation
N=600N=200
OR
N= >2000
CGIIllumina
• Development of Clinical research exome reports for the Autism Genome Project
Autism Project Design and Results
• Newfoundland cohort pilot (Bridget Fernandez) with 75 trios run on Solids 5500xL and 75 trios with Ion Proton:
• 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)
Annotated SNVs and INDELS
<1% AF in all databases?
Variant Deleterious?
Deleterious related to phenotype
Further Research, Gene Discovery, Secondary Findings
Variants of Primary interest ‐
Segregation and Genotype‐Phenotype correlation
YES
NONO
YES YES
VUS VUS related to the phenotype
De novo variant
ASD/Cognitive
YESNO
INTERPRETATION: maternally inherited NRXN1 G989* may be pathogenic variant in this family
NRXN1 exon 15 Gly989stop(chr2: 50,724,505 C>A)Neurexin 1 – nervous system cell adhesion molecule and ASD candidate gene
NRXN1 G989*
NRXN1 G989*
Inherited variants play a role in Autism
Whole Exome Sequencing:
INTERPRETATION: NRXN1 G989* may be pathogenic variant with 15q11.2 CNV also contributing to phenotype
NRXN1 exon 15 Gly989stop(chr2: 50,724,505 C>A)Neurexin 1 – nervous system cell adhesion molecule and ASD candidate gene
15q11.2 loss
15q11.2 loss
NRXN1 G989*
15q11.2 535kb loss(HERC2P2, CYFIP1, NIPA2, NIPA1, TUBGCP5, WHDC1L1, GOLGA9P)Known ASD association with variable expressivity
NRXN1 G989*
Interpretation depends on technology
Whole Exome Sequencing:
Microarray CNV analysis:
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
AcknowledgementsThe Centre for Applied GenomicsThe Hospital for Sick ChildrenStephen W. SchererLynette LauSergio PereiraBhooma ThiruvDaniele MericoSusan WalkerKristiina TammimiesRyan Yuen
Funding:
Genome Clinic ProjectRonald Cohn
Stephen MeynSarah BowdinRonald Cohn
Nasim MonfaradPeter Ray
James Stavropoulos
Life Technologies
Sickkids Clinicians Roberto MendozaTino PicisoneChristoph LitchElise Heon
Karen Keith, Matt Dyer, Yang Wang, Michael Lelivelt, Fiona Hyland, Michael Gallad, Kate Rhodes, Mathieu Lariviere
Autism Project Collaborators Peter Szatmari (McMaster)Wendy Roberts (Sickkids), John Vincent (CAMH), Bridget Fernandez (MUN), EvdokiaAnagnostou (Bloorview), Lonnie Zwaigenbaum (Univ of Alberta)
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.
Sponsored by:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
Christian Gilissen, Ph.D.Radboud University Medical Centre NijmegenThe Netherlands
Christian Marshall, Ph.D.The Hospital for Sick ChildrenToronto, Canada
Shifting the Paradigm in Translational andClinical Research
Shifting the Paradigm in Translational andClinical Research 4 December, 20134 December, 2013
Exome Sequencing in Today’s LabExome Sequencing in Today’s LabWebinar SeriesWebinar SeriesScienceScience
Exome Sequencing in Today’s Lab:Shifting the Paradigm in Translational and Clinical Research
Christian Gilissen [email protected]‐11‐2013
Human genetics Nijmegen
Research
Clinicalgenetics
Genome diagnostics
Sanger Targeted Exome Genome• Very accurate
• 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 yousequence
• 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
• Sufficient patients required
• Sequencing bias
• No non‐coding regions
• Incidental findings
• Data analysisbottleneck
• Interpretation of non‐coding variants
• Expensive, time‐consuming
Why exome sequencing?
Approaches
Variantspatient
Genepackage
Gene package approachMost genes known
Pilot study: 50 exomes for 5 disorders
Trio approachMost genes unknown
Neveling et al. Hum mut. 2013 De Ligt et al. NEJM, 2012
Pilot study: 100 trios for intellectual disability
De novo variants
Variants in known genes
Workflow
Enrichment
• Enrichment (Agilent v4)• ~ 21,000 genes
Sequencing
• Sequencing at BGI Copenhagen
• Using Illumina 2x100bp, 75x median coverage
PrimaryAnalysis
• Read mapping with BWA• Variant calling with GATK
Secondary Analysis
• Quality control• Sample mix‐up check• Variant annotation
Inter‐pretation
• Gene package visualization• Standardized interpretation protocol
• Independent interpretation by 2 people
Report
• Validation by Sanger• [Segregation analysis and functional confirmation]
• Report of results
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
0.00%10.00%20.00%30.00%40.00%50.00%60.00%70.00%80.00%90.00%
100.00%
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49
0
0.5
1
1.5
2
2.5
F F MMMMM F F MM F MM F F F M F M F F M F M F F F M F F F MMGender according to patient database (M=male, F = Female)
Sample mix‐up
• Gender check:• Calculate chrY/chrX target
coverage ratio
• SNP Test:• 12 common SNPs tested
separately by Sanger sequencing
• Trio check:• Compare high quality variant calls
between child and parents0
0.20.40.60.8
1
no swap patient -parent swap
parent -unrelated
swap
child -unrelated
swap
Coverage ratio
Variants and annotation
Qualitycontrol Filtering
Patient DB
Variant DB
How to do 400 samples per month?
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
blind deaf move mito
Number of genes(Sept. 2011)
Blindness 144
Deafness 98
Early onset colorectalcancer 115
Mitochondrial disorders 207
Movement disorders 152
Neveling et al. Hum mut. 2013
Yield
0%
10%
20%
30%
40%
50%
60%
Sanger
Exome
25%
52%
11%16%
0% 3%5%
20%
10%
44%
8%
33%
29%
0%
0%
Neveling et al. Hum mut. 2013
%Maximum % of cases solved if all available genes had been Sanger sequenced
=
Current packages
# genes in disease package
99115
144 145
219
0
100
200
300
400
500
600
131115
183
240 234
15
64
265
534563
116
2246
0
100
200
300
400
500
600
Current packages
# genes in disease package
131115
183
240 234
15
64
265
534563
116
2246
0
100
200
300
400
500
600
Current packages
Exome sequencing can be cost-efficient compared to Sanger when sequencing 3 genes or more..
# genes in disease package
131115
183
240 234
15
64
265
534563
116
2246
0
100
200
300
400
500
600
Current packages
# genes in disease package
Pilot study – de novo approach
• 100 patients + 200 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
Positive diagnosisJune 2012 June 2013
All mutations 16 29De novo mutations 13 28
Autosomal dominant 10 23X‐linked 2 4Autosomal recessive 1 1
Inherited mutations 3 1X‐linked 3 1Autosomal recessive 0 0
Candidates 19 11
Yield in 100 ID patients
Yield of ~30% in patients with severe IDDe Ligt et al. NEJM, 2012
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 5th 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 novomutations outside of the package
“Open the exome”
Nat gen. 2012
Matching phenotype
Conclusions• Exome sequencing results in a higher yield for genetically heterogeneous
diseases than Sanger‐based approaches
• De novomutations 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
Acknowledgments
ALL PATIENTS, PARENTS & CLINICIANS
WORLDWIDE!
Genome DiagnosticsHelger YntemaErik‐Jan KamsteegLies HoefslootWilly NillesenMarjolijn LigtenbergArjen MensenkampDorien LugtenbergRolph Pfundt
Clinical geneticsMarjolein WillemsenTjitske KleefstraErnie BongersDavid KoolenAnneke Vulto‐van SilfthoutWendy van Zelst‐StamsSascha Vermeer
Genome ResearchRick de ReuverMarisol del RosarioNienke WieskampThessa KroesPetra de VriesMichael KwintIrene JanssenMarloes Steehouwer
Kornelia Neveling Lisenka Vissers Alex Hoischen Joep de Ligt Ilse Feenstra Bregje van Bon
Joris Veltman Marcel Nelen Bert de Vries Han BrunnerHans Scheffer
Acknowledgments
Sponsored by:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
To submit your questions, type them into the text box
and click .Christian Gilissen, Ph.D.Radboud University Medical
Centre NijmegenThe Netherlands
Christian Marshall, Ph.D.The Hospital for Sick ChildrenToronto, Canada
Shifting the Paradigm in Translational andClinical Research
Shifting the Paradigm in Translational andClinical Research 4 December, 20134 December, 2013
Exome Sequencing in Today’s LabExome Sequencing in Today’s LabWebinar SeriesWebinar SeriesScienceScience
Look out for more webinars in the series at:webinar.sciencemag.org
For information related to this webinar, go to:www.lifetechnologies.com/ionexome
To provide feedback on this webinar, please e‐mailyour comments to [email protected]
Sponsored by:
Brought to you by the Science/AAAS Custom Publishing Office
Shifting the Paradigm in Translational andClinical Research
Shifting the Paradigm in Translational andClinical Research 4 December, 20134 December, 2013
Exome Sequencing in Today’s LabExome Sequencing in Today’s LabWebinar SeriesWebinar SeriesScienceScience