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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
4CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016
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)
12CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016
§ 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
14CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016
§ 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
15CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016
Variant Annotationand FiltrationTool
Area1
Area2 Area3
Area4
16CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016
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
17CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016
Variant Annotationand FiltrationToolArea2 FiltrationArea
Thisareaallowsdefinitionoffiltration
criteriaincluding:
"Variationtype""Frequency""Prediction”"Genesinformation""Others"
18CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016
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
29CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016
§ Indexcaseonly
§ Diagnoseddisease:Charcot-Marie-ToothDiseaseType4§ Modeofinheritance:Autosomalrecessivetransmission
(noconsanguinity)
§ SequencingPlatform:Illumina
§ Files:
§ 1annotatedfile(CMT4.ann.hg19_multianno_umd.txt)
Usecase#1
30CBéroud,JPDesvignes– VEPOctober31st – November3rd 2016
§ 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