基因組變異檢測(cè)位點(diǎn)注釋方法介紹

組變異檢測(cè)8 組：利用高通量技術(shù)，對(duì)及對(duì)照組織的DNA進(jìn)序；通過(guò)生物信息分析，與對(duì)照組織的比較，得到組織特有的單核苷酸變異，腫瘤是高度異質(zhì)性的，其中致病突變可能以極低比例存在。GATKHG模塊和SomeballparkmetricsforcancerCallsomatic Germlinevs.somaticSomaticMutationsandpassengermutations:havenodirectorindirecteffectonaselectiveadvantageoftumorDrivermutations:haveaselectivegrowthadvantageintumor78ThreemajorclassesofDNAcross-individual:evensmalllevelsofcontaminationcancausemanyfalsepositives(ContEst)within-individual：suchasnormalDNAcontaminationoftumorleadstodecreasedsensitivity -seqandcross-species: ContaminationEstimationforcross-samplejava-jar -T-R-I:eval-I:genotype-L-isr-oContaminantsitesvaryfrompatienthomozygousHowdoIknowwhichsitesarehomozygousPopulationallelesampleMuTectwashybridizedwithHaplotypeCallertocallsomaticSNVs&indelsMuTect2appliesfiltersasVCFHaplotypeCaller’sreassemblymethoddetectsbothSNVsandBuilt-infilterseliminatefalsepositives,increasingOptionalfiltersscreenforartifactsandgermlineVCFannotationsincludenewFalsepostivescomefromartifactsandgermlineWouldyouconsiderthisasomaticvariant,orsimplyanerrorpronesite?Howtorun-jar ysisTK.jar\GATK-TMuTect2-Rreference.fasta\參 -I:tumortumor.bam\腫瘤樣 -I:normalnormal.bam\正常樣 --dbsnpdbSNP.vcf\dbSNP--cosmicCOSMIC.vcf\ -Lerval_list\-o Createyourownpanelofnormals(PoN)in2stepsStep1:Createasample-levelPoNper-jar ysisTK.jar-TMuTect2-Rreference.fasta-I:tumornormal1.bam--cosmicCOSMIC.vcf-oCreateyourownpanelofnormals(PoN)in2stepsStep2.CombinedPoNretainsvariantscalledinatleasttwo-jar ysisTK.jar-Rreference.fasta-Voutput.normal1.vcf-Voutput.normal2.vcf[-Voutput.normal2.vcf...]-minN2--setKey"null"-oMuTect2’sbuilt-infiltersmarkfalsepositives,increasespecificityDownstreamhard-filtersallowupstreamsensitivityOptionalfiltersscreenfornoise&germlineIfsiteisinPoN(≥2samples),VCFFILTERfield:VCFINFOfield:PON=#,where#isthecountfromthePoNIfavariantisindbSNPbutnotinCOSMIC,VCFFILTERfield:FlaginVCFINFOfield:? :癌旁組織 深度低（duplicationreads比例高DNA 組一起callsomaticmutation會(huì)非常目前只能單線程跑，可考慮一條一 同時(shí)VarScan2samtoolsmpileup-C50-q1-B-fref.fanormal.bam>samtoolsmpileup-C50-q1-B-fref.fatumor.bam>Step2：根據(jù)normal和tumor的mpileup文件來(lái)callsomaticjava-jarVarScan.jarsomaticnormal.mpileuptumor.mpileup--output-snpout.varscan.snp--output-indelout.varscan.indel--output-vcf1Step3：對(duì)somaticsnpout.varscan.indel.vcf--output-fileout.varscan.filtered.snp.vcfjava-jarVarScan.jarprocessSomaticjava-jarVarScan.jarprocessSomatic從左至右以此分別為、位置、該位點(diǎn)的參考?jí)A基、該位點(diǎn)reads覆output.snp.Somatic.hc(high-confidenceSomaticoutput.snp.Somatic.lc(low-confidenceSomaticoutput.snp.Germline(sitescalledoutput.snp.LOH(sitescalledloss-of-heterozygosity,or .AG.PASS GT:GQ:DP:RD:AD:FREQ:DP40/1:.:18:9:9:50%:3,6,3,61/1:.:31:4:24:85.71%:2,2,13,11 .ACT A. DP=87;SS=3;SSC=32;GPV=1E0;NoticeNoticeMuTect2和VarScan2檢測(cè)到的腫瘤 (1)過(guò)濾read(2)SomaticSNP/INDELAnotationSomaticSNP/INDELAnotationVariantClassification：3'UTR;5'UTR;IGR;Intron;Missense_Mutation;Nonsense_Mutation;Silent;Frame_Shift_Del;Frame_Shift_Ins;SomaticSNP/INDELAnotation輸入格式 Singlenucleotidevariants：chr4150150AInsertions:-TDeletions:A-MAFFigure1FiveapproachestodetectCNVsandSVsfromNGSshortRunVarScancopynumberonnormalandtumormpileupsamtoolsmpileup-C50-q1-B-fref.fa-ltarget.bednormal.bamtumor.bam>java-jarVarScan.jarcopynumbernormal-tumor.pileupvarScan--mpileup1java-jarVarScan.jarcopynumbernormal.pileuptumor.pileupvarScanRunVarScancopyCallertoadjustforGCcontentandmakepreliminaryjava-jarVarScan.jarcopyCallervarScancopynumber--output-file3.ApplycircularbinarysegmentationusingtheDNAcopylibraryfromBioConductorcn<-read.table("CNA.object<-CNA(genomdat=cn[,6],chrom=cn[,1],maploc=cn[,2],data.type='logratio')CNA.smoothed segs<-segment(CNA.smoothed,verbose=0,segs2=write.table(segs2[,2:6],file="out.file",s=F,s=F,quote=F,sep="\t")--data-ratio-Thenormal/tumorinputdataratioforcopynumberplot(segs,plot(segs,OthermethodsforCNVV:ExomeSequencing-BasedCopy-NumberVariationandLossHeterozygosityCNVnator:Anapproachtodiscover,genotype,andcharacteriztypicalandatypicalCNVsfromfamilyandpopulationgenomesequencingCNVer:DetectingcopynumbervariationwithmatedshortCNVseq:CNV-seq,anewmethodtodetectcopynumbervariationusinghigh-throughputsequencing StructuralvariationsCRESTcallStep1：convertthegenomefiletoa2bitrepresentationsuitablefortheStep2：startupthegfServerstartlocalhost50000Step3：Getsoft- perlextractSClip.pl-inormal.bam--ref_genomegenome.fa-operlextractSClip.pl-itumor.bam--ref_genomegenome.fa-oStep4：RemovegermlineperlcountDiff.pl-dtumor.bam.cover-gnormal.bam.cover>CRESTcallStep5：RunningtheSVdetectionperlCREST.pl-ftumor.bam.cover.somatic.cover-dtumor.bam-gnormal.bam--ref_genomegenome.fa-o./-tgenome.2bit--blatserverlocalhost--blatport50000>crest_out.logStep6：Visulizationofthedetailedalignmentatperlbam2html.pl-dtumor.bam-gnormal.bam-iStep7：cleanupgfServerstoplocalhostCREST ,+,9, ,+,6,INS,40,42,68,64,0.9307259192758010.912801348748934,0,1, ,264,CREST ,+,9, ,+,6,INS,40,42,68,64,0.9307259192758010.912801348748934,0,1, ,264,OthermethodsforSVBreakDancer(BreakDancer:Analgorithmforhighresolutionmap genomicstructuralvariation)perlbam2cfg.plsample.bam>sample.cfgbreakdancer_maxsample.cfg>raw_SV.txtPindel(Pindel:apatterngrowthapproachtodetectbreakpointsoflargedeletionsandmediumsizedinsertionsfrompaired-endshortreadspindel-fref.fa-iconfig.txt-opindel-cALL-T20-s-l-b Mutationfrequency-basedPrinciple：passengermutationsarelikelytoberandomlydistributedacrossthegenome,whereasdrivermutationsclusteraroundcancergenes(clusterinsoraroundspecificaminoFunctionalimpact-basedPrinciple:primarilyrelyonestimatingthedeleteriouseffectsofSNVsviaevaluatingaminoacidconservationatthecorrespondingpositions.PolyPhen-Structuralgenomics-basedPrinciple:themutationsatthestructurallyimportantsitesaremorelikelylinkedtodiseaseordrugtargets.Thesesitesincludespecificproteinregions(e.g.protein ,intrinsicallydisorderedregions),posttranslationalmodification(PTM)sites(e.g.phosphorylationsites),proteinpocketsandprotein–ligandbindingsites.Network-orpathway-basedPrinciple:cancerisacomplexdisease,withmanychangesalteredatthenetworkandpath-waylevels,notsimplyapointmutation.Dataintegration-basedBackground:Cancer‘panomics’data,includingsomaticmutations,transcriptome,methylationandpro