Illumina测序介绍

©2013Illumina,Inc.Allrightsreserved.Illumina,IlluminaDx,BaseSpace,BeadArray,BeadXpress,cBot,CSPro,DASL,DesignStudio,Eco,GAIIx,GeneticEnergy,GenomeAnalyzer,GenomeStudio,GoldenGate,HiScan,HiSeq,Infinium,iSelect,MiSeq,Nextera,NuPCR,SeqMonitor,Solexa,TruSeq,TruSight,VeraCode,thepumpkinorangecolor,andtheGeneticEnergystreamingbasesdesignaretrademarksorregisteredtrademarksofIllumina,Inc.Allotherbrandsandnamescontainedhereinarethepropertyoftheirrespectiveowners.Part#15045845_Rev.CIllumina测序技术概述2Part#15045845_Rev.CFORRESEARCHUSEONLY课程结束时：–清楚了解Illumina测序流程中的主要步骤–能够描述簇生成（ClusterGeneration）的过程–理解边合成边测序（SequencingbySynthesis）的原理课程目标3Part#15045845_Rev.CFORRESEARCHUSEONLYIlluminaSequencingWorkflowLibraryPreparationcBotMiSeqNextSeqHiSeq2500-RapidClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeqSequencingDataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析4Part#15045845_Rev.CFORRESEARCHUSEONLYIlluminaSequencingWorkflowLibraryPreparationcBotMiSeqNextSeqHiSeq2500-RapidClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeqSequencingDataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析5Part#15045845_Rev.CFORRESEARCHUSEONLY文库制备的目的是在需要测序的DNA片段两端加上能够与测序仪配合的接头序列（Multiplexed,SR,PE）双端标签文库文库制备是决定测序实验成功与否的关键步骤①①①与流动槽（FlowCell）结合的区域②②②Read1和Read2测序引物结合的区域③③插入片段④④④标签序列区域（Index）6Part#15045845_Rev.CFORRESEARCHUSEONLYIlluminaSequencingWorkflowLibraryPreparationcBotMiSeqNextSeqHiSeq2500-RapidClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeqSequencingDataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析7Part#15045845_Rev.CFORRESEARCHUSEONLYWhatisaFlowCell?Eachlaneisrandomlycoatedwithalawnofoligosthatarecomplementarytolibraryadapters每条通道中都随机植入了能与文库接头互补结合的大量短DNA片段Clustergenerationoccursonaflowcell簇生成在流动槽（flowcell）上完成流动槽(FlowCell)是什么?Aflowcellisathickglassslidewithchannelsorlanes一种含有通道的厚玻璃片8Part#15045845_Rev.CFORRESEARCHUSEONLY簇生成SingleDNALibrary单个DNA文库分子AmplifiedClonalCluster扩增后的DNA克隆簇cBotSequencer9Part#15045845_Rev.CFORRESEARCHUSEONLYHybridizeFragment&ExtendAdaptersequence接头序列3’extension3’延伸Surfaceofflowcellcoatedwithalawnofoligopairs流动槽表面随机植入了大量引物链SingleDNAlibrariesarehybridizedtoprimerlawn变性后单链DNA文库与流动槽上的引物杂交Boundlibrariesarethenextendedbypolymerases随后在DNA合成酶的作用下，结合上的DNA文库进行延伸片段杂交与延伸10Part#15045845_Rev.CFORRESEARCHUSEONLYNewlysynthesizedstrand新合成链Originaltemplate原始模板链DenatureDouble-StrandedDNADiscard洗去丢弃Double-strandedmoleculeisdenatured双链DNA分子被变性Originaltemplatewashedaway原始的模板链被洗去Newlysynthesizedstrandiscovalentlyattachedtoflowcellsurface新合成的DNA链以共价键连接的方式结合在流动槽表面双链DNA变性11Part#15045845_Rev.CFORRESEARCHUSEONLYNOTE:SinglemoleculesbindtoflowcellinarandompatternNOTE:单个DNA分子以随机的方式与流动槽表面结合Single-StrandedDNA单链DNA12Part#15045845_Rev.CFORRESEARCHUSEONLYBridgeAmplificationSingle-strandedmoleculeflipsoverandformsabridgebyhybridizingtoadjacent,complementaryprimer共价键结合在流动槽表面的单链DNA分子与其附近的互补引物杂交，整条DNA分子折叠后形成一种类似于桥的结构。Hybridizedprimerisextendedbypolymerases在DNA合成酶作用下，杂交后的引物以单链DNA为模板进行延伸桥式PCR扩增3’extension3‘延伸反应13Part#15045845_Rev.CFORRESEARCHUSEONLYBridgeAmplificationDouble-strandedbridgeisformed延伸完成后形成双链DNA桥式结构桥式PCR扩增14Part#15045845_Rev.CFORRESEARCHUSEONLYDenatureDouble-StrandedBridgeDouble-strandedbridgeisdenatured桥式结构的双链DNA被变性Result:Twocopiesofcovalentlyboundsingle-strandedtemplates结果：形成两条与流动槽表面共价键结合的DNA模板双链DNA桥式结构变性15Part#15045845_Rev.CFORRESEARCHUSEONLYBridgeAmplificationSingle-strandedmoleculesflipovertohybridizetoadjacentprimers单链DNA分子再一次折叠后与附近的引物杂交结合Hybridizedprimerisextendedbypolymerase杂交后的引物再次在DNA合成酶的作用下延伸桥式PCR扩增16Part#15045845_Rev.CFORRESEARCHUSEONLYBridgeAmplificationBridgeamplificationcycleisrepeateduntilmultiplebridgesareformed桥式扩增不断重复发生直到形成数量足够的DNA桥（与PCR反应类似，区别在于引物不是游离在溶液中，而是固定在流动槽表面）桥式PCR扩增17Part#15045845_Rev.CFORRESEARCHUSEONLYLinearizationdsDNAbridgesaredenatured双链DNA变性后，解开桥式结构，变成线性化的单链DNA线性化18Part#15045845_Rev.CFORRESEARCHUSEONLYReverseStrandCleavageReversestrandsarecleavedandwashedaway,leavingaclusterwithforwardstrandsonly与流动槽表面结合的DNA反链被切除并洗去，只留下正链，形成包含均一单链的DNA簇反链切除19Part#15045845_Rev.CFORRESEARCHUSEONLYBlockingFree3’endsareblockedtopreventunwantedDNApriming为了防止后续测序过程中不必要的DNA延伸，对流动槽上结合的所有DNA分子的3’端进行封闭DNA链封闭20Part#15045845_Rev.CFORRESEARCHUSEONLYRead1PrimerHybridizationSequencingprimer测序引物Sequencingprimerishybridizedtoadaptersequence将Read1测序引物加入流动槽，使其与待测DNA分子的接头序列结合Read1引物杂交21Part#15045845_Rev.CFORRESEARCHUSEONLYIlluminaSequencingWorkflowLibraryPreparationcBotMiSeqNextSeqHiSeq2500-RapidClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeqSequencingDataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析22Part#15045845_Rev.CFORRESEARCHUSEONLYSequencingBySynthesisAdd4Fl-NTP’s+Polymerase加入4种不同荧光标记的dNTP和DNA合成酶IncorporatedFI-NTPimaged对结合上的荧光dNTP进行照相Terminator&fluorescentdyecleavedfromFI-NTP结合在DNA链上的荧光NTP中的荧光标记和阻断基团被切除，可以继续下一轮反应X36-251边合成边测序（SBS）23Part#15045845_Rev.CFORRESEARCHUSEONLY•All4labelednucleotidesin1reaction同时存在4种不同标记的核苷酸•Higheraccuracy更高的准确性•Noproblemswithhomopolymerrepeats不存在均聚物重复片段测序困难问题•Incorporation结合•Detection检测•Deblock去阻断•FluorRemoval切除荧光基团NextCycle下一个循环ReversibleTerminatorChemistry可逆阻断3‘阻断基团切除位点荧光基团去阻断后自由的3‘OH端