VarAFT and UMD -Predictor - Amazon S3 · VarAFT and UMD -Predictor training course Variant Effect...

1CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

VarAFT and UMD-Predictortraining course

VariantEffectPredictionTrainingCourseHeraklion,Greece,2016

Jean-PierreDesvignes&ChristopheBéroud,

Aix-MarseilleUniversity

INSERMUMR_S910MedicalGeneticsandFunctionalGenomics

2CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

UMD-Predictor: A pathogenicity prediction system for cDNA SNP

VariantEffectPredictionTrainingCourseHeraklion,Greece,2016

Jean-PierreDesvignes&ChristopheBéroud,

Aix-MarseilleUniversity

INSERMUMR_S910MedicalGeneticsandFunctionalGenomics

3CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Introduction

§ GetanUMD-Predictoraccount

§ http://umd-predictor.eu

§ Providearealemailaddress– alink

toactivateyouraccountissentto

thisemailaddress

§ Locateyourtokenforwebservices

option

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HandsonUMD-Predictor

Usingthewebinterface

§ Getpathogenicitypredictionforthesespecificmutations

§ BRCA2 c.7235C>T

§ NR3C1 (NP_000167.1)p.Arg477Ser;p.Tyr478Cys;p.Leu672Pro

§ ENST00000409582c.4003A>T;ENST00000250003c.874G>T;

§ Chr10:g.103531220G>A;Chr10:g.103530356C>T

5CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

HandsonUMD-Predictor

Usingthewebinterface

§ Getpathogenicitypredictionforthesespecificmutations

§ BRCA2 c.7235C>T

6CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

HandsonUMD-Predictor

Usingthewebinterface

§ Getpathogenicitypredictionforthesespecificmutations

§ NR3C1 (NP_000167.1)p.Arg477Ser;p.Tyr478Cys;p.Leu672Pro

7CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

HandsonUMD-Predictor

Usingthewebinterface

§ Getpathogenicitypredictionforthesespecificmutations

§ ENST00000409582c.4003A>T;ENST00000250003c.874G>T;

8CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

HandsonUMD-Predictor

Usingthewebinterface

§ Getpathogenicitypredictionforthesespecificmutations

§ Chr10:g.103531220G>A;Chr10:g.103530356C>T

9CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

VarAFT: A variant annotation and filtration system for next generation

sequencing data

VariantEffectPredictionTrainingCourseHeraklion,Greece,2016

Jean-PierreDesvignes&ChristopheBéroud,

Aix-MarseilleUniversity

INSERMUMR_S910MedicalGeneticsandFunctionalGenomics

10CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Introduction

§ NGStechnologiesgeneratealotofdata

§ WholeExome,GenesPanelandWholeGenomearenowusedtoidentify

newgenesresponsibleforhumangeneticdiseases

§ Bioinformaticspipelines(RawData� VCF)toidentifymutationshave

beengreatlyimproved

§ AnnotationandFilteringstepsneedtobeimproved

§ Only20at30%ofclinicaldiagnosiscasesweresuccessful

11CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Introduction

§ Varioussystemshavebeenreleased

§ VCF-Miner,TREAT,BierApp,Gemini,FMFilter…

§ Commandlinesystems(bioinformaticians)

§ Incompletetools(lackofannotation,lackofoption)

§ Commercialtools

§ Wedecidedtodevelopafreelyavailabletool:VarAFT.

§ VarAFTv2hasbeenreleasedonJune10th2016(freshrelease)

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§ http://varaft.eu§ standalonemultiplatformapplication§ easytouseuserinterface

§ experiments’quality§ annotation§ combination andfiltration ofdatastoredin

VCFfilesfrommultiplesamples

§ AR,AD,X-linkedorY-linked

§ PopulationGenetics

§ Cancers

§ annotations from UMD-PredictorandHSF§ optimalselectionofcandidatepathogenic

mutations

Variant Annotationand FiltrationTool

13CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

§ ImplementationofANNOVAR system

§ Oneclickdownloadmoduletogetall

neededdatafilesforannotation

§ Hg19

§ Hg38

§ AnnotationofVCF,mergedVCF,gVCFortabfile

§ IncludeUMD-Predictor scorethroughthewebservice

§ Multithreaded process.

§ ExomeAnnotation(100,000variants)

� 5-10min

§ GenomeAnnotation(4millions)

� 30-45min

Variant Annotationand FiltrationTool

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§ Listofavailableannotation§ GeneAnnotation

§ RefSeq,Ensembl

§ Frequency§ dbSNP144(orpreviousversions),ExAC,ExomevariantServer6500,

1000 Genomes,Kaviar(~KnownVARiants),HRCR(TheHaplotype

ReferenceConsortium)

§ PredictionandConservationScores§ SIFT,Polyphen2,LRT,MutationTaster,MutationAssessor,CADD,DANN,

Provean,GERP++,PhyloP,SiPhy,phastCons

§ Publicdatabase§ Clinvar,Cosmic

§ Genevarianttolerancescores§ RVIS,GDI,LofTool

Variant Annotationand FiltrationTool

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Variant Annotationand FiltrationTool

Area1

Area2 Area3

Area4

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Variant Annotationand FiltrationToolArea1

Itispossibletocombinedatafrommultiplesamples/patients(singleton,trioorany

combination)

• AutosomalRecessiveDisease

• IndexCaseonly

• TrioAnalysisbothParents/Proband

• MultiAnalysis(VCFmerged)

• AutosomalDominantDisease

• Indexonly

• TrioAnalysiswithbothParents/Proband(fordenovoanalysis)

• Customanalysis

• Commonvariants

• Commongenes

• Possibilitytosetathreshold(4outof6samples)

Selectionofcombinationmode

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Variant Annotationand FiltrationToolArea2 FiltrationArea

Thisareaallowsdefinitionoffiltration

criteriaincluding:

"Variationtype""Frequency""Prediction”"Genesinformation""Others"

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Variant Annotationand FiltrationToolArea2

⚠ Populationfrequency(notalldatabasesgatherhealthyindividuals,ethnicity)⚠ presenceindbSNPdoesnotmean“polymorphism”

FiltrationArea

Thisareaallowsdefinitionoffiltration

criteriaincluding:

"Variationtype""Frequency""Prediction”"Genesinformation""Others"

19CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Variant Annotationand FiltrationToolArea2 FiltrationArea

Thisareaallowsdefinitionoffiltration

criteriaincluding:

"Variationtype""Frequency""Prediction”"Genesinformation""Others"

20CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Variant Annotationand FiltrationToolDiscrepanciesbetweenpredictors

Venndiagramofpathogenicitypredictions of5,000variantsfromUniprotusingCADD(Kircheretal.2014),SIFT

(Simetal.2012),Polyphen2(PPH2)(Adzhubeietal.2010),Provean,MutationTaster(MutTaster)(Schwarzetal.

2014) andUMD-Predictor(UMD-Pred)(Salgadoetal.2016) pathogenicitypredictionsystems

⚠ Combiningpredictorswill

addalotoffalse(+/-)

⚠ Canremoveavariantof

interestifnotwellpredicted

byoneofthose

21CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Variant Annotationand FiltrationToolArea2 FiltrationArea

Thisareaallowsdefinitionoffiltration

criteriaincluding:

"Variationtype""Frequency""Prediction”"Genesinformation""Others"

22CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Variant Annotationand FiltrationToolArea2

Youcansaveandre-applyfiltrationoptions

FiltrationArea

Thisareaallowsdefinitionoffiltration

criteriaincluding:

"Variationtype""Frequency""Prediction”"Genesinformation""Others"

23CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Variant Annotationand FiltrationToolArea3 InformationArea

Thisareaprovidesdetailedinformationontheselectedvariant:

"Nomenclatureinalltranscripts""VariantsInformationforeachsamples""Frequencyingeneralpopulation""PathogenicityPredictions""Localizationinparticularregions""Pathways(KEGG,REACTOME,PID)""GeneOntology""OMIM""TissuesExpressionfromGTEX""Externallinks"

24CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Variant Annotationand FiltrationTool

Listofselectedvariationswithmainannotations.Aclickonarowwillupdatearea#3.

A rightclickgivesaccesstoapopupmenuwhichallowsto:

- accesstoIGV andvisualizemappingdata

- showtissuesexpressions- getdatafromHSF- accessseverallinks(NCI,UCSC,GeneCards…)

Theusercanchoosewhichdatatodisplayinthetable

Area4 Resultstable

25CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

CoverageAnalysis

• ImplementationofBEDtools

• Wholeexome/genepanel/custombed

• HG19/HG38

• Userfriendlyinterfacetovisualizecoverageresults

• Interactivecharts

• Visualizationforonetranscriptorthewholegene

• Exportresultsasareporttoclassifygene

Variant Annotationand FiltrationTool

26CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

CoverageAnalysis

Variant Annotationand FiltrationTool

27CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

§ VarAFTisanuserfriendlytoolthatallowstheannotation,filtrationandthecoverageanalysisofNGSdata

§ Itisroutinelyusedforclinicaltestingandresearch§ ItwasusedduringjamboreeorganizedbytheEuropeanRD-Connectproject§ ItisusedbyresearchersfromtheUK10K

RecentpublicationssuccessfullyusedVarAFTtoperformthevariantprioritization:

§ Gorokhova,S. etal. Comparingtargetedexomeandwholeexomeapproachesforgeneticdiagnosisofneuromusculardisorders.Applied &TranslationalGenomics 7, 26–31(2015).

§ Rapetti-Mauss,R. etal. AmutationintheGardos channelisassociatedwithhereditaryxerocytosis. Blood 126, 1273–1280(2015).

§ Lacoste,C. etal. Coverageanalysisoflistsofgenesinvolvedinheterogeneousgeneticdiseasesfollowingbenchtopexomesequencingusingtheionproton. JGenet 95, 203–208(2016).

§ Galant,D. etal. AHeterozygousZMPSTE24MutationAssociatedwithSevereMetabolicSyndrome,EctopicFatAccumulation,andDilatedCardiomyopathy. Cells 5, 21(2016)

Conclusion

28CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

§ Datafrom

§ Krawitz PM,MurakamiY,HechtJ,KrugerU,HolderSE,etal.(2012)

MutationsinPIGO,amemberoftheGPI-anchor-synthesispathway,

causehyperphosphatasia withmentalretardation.AmJHumGenet91:

146–151.doi:10.1016/j.ajhg.2012.05.004

§ MergedVCFwithbothparentsand2affecteddaughters§ Autosomalrecessivetransmission(noconsanguinity)§ AffectedDaughters:464,465§ Mother:466§ Father:467

Demonstration

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§ Indexcaseonly

§ Diagnoseddisease:Charcot-Marie-ToothDiseaseType4§ Modeofinheritance:Autosomalrecessivetransmission

(noconsanguinity)

§ SequencingPlatform:Illumina

§ Files:

§ 1annotatedfile(CMT4.ann.hg19_multianno_umd.txt)

Usecase#1

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§ CompoundheterozygousmutationintheSH3TC2 gene

§ c.279G>A(p.K93K)

§ c.2860C>T(p.R954X)

§ UseOMIM andHSF

Usecase#1:result

31CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Usecase#2

§ Datatype:trio [parents+1affectedchild]

§ Diagnoseddisease:Limb-GirdleMuscularDystrophy§ Modeofinheritance:Autosomalrecessivetransmission

(Nodataaboutconsanguinity)

§ SequencingPlatform:Illumina

§ Experimenttype:Genepanelsequencing

§ 4Files:

§ 1annotatedfilefortheaffectedchild

§ 1annotatedfileforthemother

§ 1annotatedfileforthefather

§ +BAMfiles

32CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

§ CompoundheterozygousmutationintheCAPN3gene

§ c.848C>T(p.M283T)

§ c.1611C>A(p.Y537X)

§ UseOMIMandliterature

§ LookatthecoverageoptionusingtheBAMfile

Usecase#2:result

33CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016

Usecase#2:CAPN3 coverage