exome sequencing covers >98% of mutations identified on targeted next generation sequencing panels 2017 holly laduca学术.pdf

该【exome sequencing covers >98% of mutations identified on targeted next generation sequencing panels 2017 holly laduca学术 】是由【周瑞】上传分享，文档一共【11】页，该文档可以免费在线阅读，需要了解更多关于【exome sequencing covers >98% of mutations identified on targeted next generation sequencing panels 2017 holly laduca学术 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..RESEARCHARTICLEExomesequencingcovers>98%ofmutationsidentifiedontargetednextgenerationsequencingpanelsHollyLaDuca1*,*,HuyVuong1,Hsiao-MeiLu1,WenboMu1,LaylaShahmirzadi1,ShaTang1,JeffereyChen1,ShrutiBhide1,,ics,AlisoViejo,California,UnitedStatesofAmerica,2DepartmentofPediatrics,icsandGenomicMedicine,UniversityofCaliforniaIrvine,Irvine,a1111111111California,UnitedStatesofAmericaa1111111111a1111111111*hladuca@(HL);kfarwell@(KDF)a1111111111a1111111111AbstractESSBackgroundCitation:LaDucaH,FarwellKD,VuongH,LuH-M,Withtheexpandedavailabilityofnextgenerationsequencing(NGS)-icMuW,ShahmirzadiL,etal.(2017)Exometests,cliniciansseekingtotestpatientswithMendeliandiseasesmustweighthesuperiorsequencingcovers>98%(WES)whenconsideringtheirfirst-,weusePLoSONE12(2)::.0170843aninsilicoanalysistopredicttheanalyticsensitivityofWESusingpathogenicvariantsiden-:NoamShomron,TelAvivUniversity,ISRAELReceived:September1,2016MethodsAccepted:January11,2017Correspondingnucleotidepositionsfor1533differentalterationsclassifiedaspathogenicorPublished:February2,2017likelypathogenicidentifiedontargetedNGSmulti-genepaneltestsinourlaboratorywereinterrogatedindatafrom100randomly-selectedclinicalWESsamplestoquantifytheCopyright:?,whichinhereditarycancer,X-linkedintellectualdisability,primaryciliarydyskinesia,Marfansyn-permitsunrestricteduse,distribution,anddrome/aorticaneurysms,,:AllrelevantdataarewithinthepaperanditsSupportingInformationWhenassessingcoverageamong100individualWESsamplesforeachpathogenicvariantfiles.(153,300individualassessments),%(n=152,798)wouldlikelyhavebeendetectedonFunding:,withaformofsalariesforallauthors,%(n=1491)-anyadditionalroleinthestudydesign,datagenicvariants,,decisiontopublish,-rich,repetitive,,CompetingInterests:Allauthorswerefulltimeicsatthetimetheasimilaranalysiswasperformedagainstcoveragedatafrom60,(ExAC).ResultsfromthisvalidationconfirmedPLOSONE|DOI:.0170843February2,20171/11:..%(91,743,296/93,062,298)-(NGS)-ictests,cliniciansarefacedwiththedecisiontopursuetargetedgenepanelsversuswholeexomesequencing(WES)astheirfirst-tiertestingapproach[1].,sequence-specificenrichmentisperformedpriortoNGStohelpachievehighcoverageandreduceoreliminatelow-coverageregions(typically~10-50X).Commonly,SangersequencingisappliedtoregionswhicharerecalcitranttoNGSenquiry,duetotechnicalorbiologicallimitations,includingGC-,currentestimatesofcoverageachievedfromwholeexomecaptureandsequencingare90±95%at>20X,withfactorssuchastargetenrichmentdesign,off-targetcapture,repetitiveandGC-orAT-richregions,copy-numbervariations,andstructuralvaria-pletecapture[2±5].UnlikeNGSpanels,theadditionofSangersequencingforregionswithinadequatecoveragewouldnotbetimeorcost-effectiveforWES,wheremorethan20,,,,amongpositiveWESfindings,23%arewithingenescharacterizedwithinthelasttwoyearsand7%arenovelgenediscoveries[6].Assuch,anadvantageofWESistheabilitytosequencetheentireexomeatonce,allowingfortheanalysisandinterpretationofallalterationsinbothwellcharacterizedandnovelgenes,andalsoallowingforre-icassocia-,thepotentialtoidentifynovelgenes,andtheabilitytosequencetheexomesofmultiplefamilymemberssimul--age,asrelatedtoaspecificgeneorsetofdiagnosticgenes[7].Here,(AlisoViejo,CA)wasqueriedforallalterationsclas-sifiedaspathogenicandlikelypathogenic(hereinreferredtocollectivelyas`pathogenicvari-ants')argetedNGSmulti-genepaneltestingofover50,|DOI:.0170843February2,20172/11:..-tieredclassificationalgorithm[8]icsandGenomicsandtheInternationalAgencyforResearchonCancer[9,10].,largeinsertions,andlargedeletionsdetectedonarrayormultiplexligation-dependentprobeamplification--genepanelstargetedarangeofhereditary(Mendelian)disordersincludingcancersusceptibility,X-linkedintellectualdisability(XLID),primaryciliarydyskinesia(PCD),Marfansyndrome,thoracicaorticaneurysmsanddissections,andrelateddisorders(Marfan/TAAD),andothercardiovasculardiseasessuchascardiomyopathiesandarrhythmias,andwereselectedbytheorderingclinicianbasedonthepatients'-domly-icsforclinicalWES,,andfordeletionsandindelscoveragewasassessedforthefirstandlastnucleotides,withthelowerofthetwoval-,thisresearchwasdeter-(SolutionsInstitutionalReviewBoard,ReferenceNumber1OCT14-93).NGSpanelsequencingLibrarypreparation,sequencing,bioinformatics,anddataanalysiswereperformedasprevi-ouslydescribed[8,11].Briefly,sampleswereenrichedforsequencetargetsusingRaindanceThunderstormtechnology(RainDanceTechnologies,Billerica,MA),andsequencedusingpaired-end,100-cyclechemistryontheIlluminaHiSeq2000(Illumina,SanDiego,CA).Importantly,PCRduplicatedsequenceswereremovedfromthedatasetpriortoalignment,(GRCh37)andvariantcallsweregeneratedusingCASAVAandPindel[12].No/low-coverageregions(.<50X)andvariantcallsotherthanknownnon-pathogenicalter-,sequencing,bioinformatics,anddataanalysiswereperformedaspreviouslydescribed[6,13,14].Briefly,(RocheNimbleGen,Madison,WI)andsequencedusingpaired-end,100-cyclechemistryontheIlluminaHiSeq2000(Illumina,SanDiego,CA).Thesequencedatawerealignedtothereferencehumangenome(GRCh37)[6].Briefly,stepwisefilteringincludedtheremovalofvariantswithqualityscores<20andallelecounts<monSNPs,intergenicand3'/5'UTRvari-ants,non-splice-relatedintronicvariants,-(AVA)[8].Allsampleswererequiredtomeetminimumqualitystandards,withatleast90%ofbasescoveredat10XandhavingbasecallqualityscoresQ20,whichtranslatestoabase-callingerrorrateof1:>|DOI:.0170843February2,20173/11:..-genepaneltest-ingwereincludedinthisanalysis,representing91genesimplicatedin5diseasecategoriesidentifiedthroughanalysisofgreaterthan~100,000alleles(S1Table).%ofpathogenicvariantsanalyzed(n=1350),ountingfor<4%(n=665,%),followedbysmalldeletions(n=485,%),intronicvariants(n=184,%),smallduplicationsandinsertions(n=169,%),andindels(n=30,%).Consideringthatcoveragewasassessedamong100individualWESsamplesforeachpatho-genicvariant(153,300individualassessments),%(n=152,798)ofpathogenicvariants(Table1).ThepercentageofpathogenicvariantswithadequatedepthfordetectiononWESwashighestamongMarfan/TAAD(%)andlowestamongXLID(%)%(n=1491)(%)andlowestamongXLID(%),genesinvolvedinXLIDrepresentedthesmall-estnumberofpathogenicvariants(n=23).%(±),andtheaveragedepthpersamplewas94X(range80X-114X)(S2Table).Furthermore,amongthese100WESsamples,98%baseswerecovered>20X,48%baseswerecovered>100Xand0%;however,therewere42pathogenicvariants(%)thatwerecovered<(%)wereinGC-richregions(definedasGC-content>60%),8wereinrepetitiveregions(%)(definedaspolymerstretching9basepairs),and3(%)(%),therewasnoobviousexplanationforno/lowexomecoverageattherespectivenucleotideposition(Table2).Thepathogenicvariantwiththelowestlevelofcoverage-(3)inPMS2,agenewithhighpseudogenehomology-wasdetectablein35ofthe100WESsam--samplevariabilitywasobservedregardingthenumberofpathogenicvariantsnotcoveredat,themediannumberofvariantslackingadequatecoverage(<10X)ineachsamplewas5(range0±12).Coverageforallpathogenicvariantspersampleispro