Illumina-官方介绍不同机型index读取方式-indexed-sequencing-over

IndexedSequencingOverviewGuideIntroduction3Single-IndexedSequencingOverview3Dual-IndexedSequencingOverview4Dual-IndexedWorkflowonaPaired-EndFlowCell4Dual-IndexedWorkflowonaSingle-ReadFlowCell6SequencingPrimersforHiSeqSystems8RevisionHistory9TechnicalAssistance11Document#15057455v03February2017ILLUMINAPROPRIETARYForResearchUseOnly.Notforuseindiagnosticprocedures.ThisdocumentanditscontentsareproprietarytoIllumina,Inc.anditsaffiliates(Illumina),andareintendedsolelyforthecontractualuseofitscustomerinconnectionwiththeuseoftheproduct(s)describedhereinandfornootherpurpose.Thisdocumentanditscontentsshallnotbeusedordistributedforanyotherpurposeand/orotherwisecommunicated,disclosed,orreproducedinanywaywhatsoeverwithoutthepriorwrittenconsentofIllumina.Illuminadoesnotconveyanylicenseunderitspatent,trademark,copyright,orcommon-lawrightsnorsimilarrightsofanythirdpartiesbythisdocument.Theinstructionsinthisdocumentmustbestrictlyandexplicitlyfollowedbyqualifiedandproperlytrainedpersonnelinordertoensuretheproperandsafeuseoftheproduct(s)describedherein.Allofthecontentsofthisdocumentmustbefullyreadandunderstoodpriortousingsuchproduct(s).FAILURETOCOMPLETELYREADANDEXPLICITLYFOLLOWALLOFTHEINSTRUCTIONSCONTAINEDHEREINMAYRESULTINDAMAGETOTHEPRODUCT(S),INJURYTOPERSONS,INCLUDINGTOUSERSOROTHERS,ANDDAMAGETOOTHERPROPERTY.ILLUMINADOESNOTASSUMEANYLIABILITYARISINGOUTOFTHEIMPROPERUSEOFTHEPRODUCT(S)DESCRIBEDHEREIN(INCLUDINGPARTSTHEREOFORSOFTWARE).©2017Illumina,Inc.Allrightsreserved.Illumina,BaseSpace,HiSeq,MiniSeq,MiSeq,Nextera,NextSeq,NovaSeq,TruSeq,thepumpkinorangecolor,andthestreamingbasesdesignaretrademarksofIllumina,Inc.and/oritsaffiliate(s)intheU.S.and/orothercountries.Allothernames,logos,andothertrademarksarethepropertyoftheirrespectiveowners.Document#15057455v03ForResearchUseOnly.Notforuseindiagnosticprocedures.2IndexedSequencingOverviewGuideIntroductionThisguideprovidesanoverviewofindexedsequencingforallIlluminasequencingsystems.Indexedsequencingisamethodthatallowsmultiplelibrariestobepooledandsequencedtogether.Indexinglibrariesrequirestheadditionofauniqueidentifier,orindexsequence,toDNA samplesduringlibrarypreparation.BaseSpaceSequenceHub,LocalRunManager,orstandalonebcl2fastq2processthesetagstoidentifyeachuniquelytaggedlibraryfordownstreamanalysis.SingleandDualIndexingThenumberofindexsequencesaddedtosamplesdiffersforsingle-indexedanddual-indexedsequencing.uSingle-indexedlibraries—Addsupto48unique6-baseIndex1(i7)sequencestogenerateupto48uniquelytaggedlibraries.uDual-indexedlibraries—Addsupto24unique8-baseIndex1(i7)sequencesandupto16unique8-baseIndex2(i5)sequencestogenerateupto384uniquelytaggedlibraries.Duringindexedsequencing,theindexissequencedinaseparateread,calledtheIndexRead,whereanewsequencingprimerisannealed.Whenlibrariesaredual-indexed,thesequencingrunincludes2additionalreads,calledtheIndex1ReadandIndex2Read.Single-IndexedSequencingOverviewThesingle-indexedsequencingworkflowappliestoallIlluminasequencingplatforms,whereanIndexReadfollowsRead1.Figure1Single-IndexedSequencing1Read 1—Read 1followsthestandardRead1sequencingprotocolusingSBSreagents.TheRead1sequencingprimerisannealedtothetemplatestrandduringtheclustergenerationstep.2IndexReadpreparation—TheRead 1productisremovedandtheIndex1(i7)sequencingprimerisannealedtothesametemplatestrand,producingtheIndex1(i7)Read.3Index1(i7)Read—FollowingIndexReadpreparation,theIndex1(i7)Readisperformed.Thereadlengthdependsonthesystemandrunparameters.Document#15057455v03ForResearchUseOnly.Notforuseindiagnosticprocedures.3IndexedSequencingOverviewGuide4Read 2resynthesis—TheIndexReadproductisremovedandtheoriginaltemplatestrandisusedtoregeneratethecomplementarystrand.Then,theoriginaltemplatestrandisremovedtoallowhybridizationoftheRead2sequencingprimer.5Read 2—Read 2followsthestandardpaired-endsequencingprotocolusingSBS reagents.Dual-IndexedSequencingOverviewDual-indexedsequencingincludes2indexreadsafterRead1:theIndex1ReadandtheIndex2Read.SequencingkitsforHiSeq®systemsareavailablewithasingle-readorpaired-endflowcell.Forallothersystems,sequencingkitsincludeapaired-endflowcell.Dual-IndexingWorkflowsThecontrolsoftwareperformsRead1,anyindexreads,andthenRead2basedontheparametersprovidedfortheruninthesamplesheetorduringrunsetup.Forallindexingworkflows,theIndex1ReaddirectlyfollowsRead1.However,fordual-indexingonapaired-endflowcell,therestoftheworkflowdiffers:uWorkflowA—TheIndex2ReadisperformedbeforeRead2resynthesis,sotheIndex2(i5)adapterissequencedontheforwardstrand.uWorkflowB—TheIndex2ReadisperformedafterRead2resynthesis,whichcreatesthereversecomplementoftheIndex2(i5)indexadaptersequence.WorkflowAWorkflowBRead1IndexReadpreparationIndex1ReadIndex2ReadRead2resynthesisRead2Read1IndexReadpreparationIndex1ReadRead2resynthesisIndex2ReadRead2preparationRead2Table1Dual-IndexPaired-EndSequencingWorkflowsDual-IndexedWorkflowonaPaired-EndFlowCellDual-indexsequencingonapaired-endflowcellfollows1of2workflows,dependingonthesystem:uWorkflowAisperformedontheNovaSeq™,MiSeq®,HiSeq2500,andHiSeq2000.uWorkflowB isperformedont