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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 4 December, 20134 December, 2013


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

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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

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Cost per Human Genome

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$ 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

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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:



Christian Gilissen, Ph.D.Radboud University Medical Centre NijmegenThe Netherlands





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

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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

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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

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Current packages


131115

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Current packages

Exome sequencing can be cost-efficient compared to Sanger when sequencing 3 genes or more..


131115

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Current packages


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:



To submit your questions, type them into the text box

and click .Christian Gilissen, Ph.D.Radboud University Medical

Centre NijmegenThe Netherlands





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]

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