Illumina测序介绍-课件

Illumina测序技术概述Illumina测序技术概述1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）的原理课程目标课程目标IlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析IlluminaSequencingWorkflow1LIlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析IlluminaSequencingWorkflow1L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②②②

Read1和Read2测序引物结合的区域③③

插入片段④④④

标签序列区域（Index）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IlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析IlluminaSequencingWorkflow1LWhatisaFlowCell?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WhatisaFlowCell?Eachlane簇生成SingleDNALibrary单个DNA文库分子AmplifiedClonalCluster扩增后的DNA克隆簇cBotSequencer簇生成SingleDNALibraryAmplifiedHybridizeFragment&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HybridizeFragment&ExtendAdaNewlysynthesizedstrand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NewlysynthesizedstrandOriginNOTE:SinglemoleculesbindtoflowcellinarandompatternNOTE:单个DNA分子以随机的方式与流动槽表面结合Single-StrandedDNA单链DNANOTE:Single-StrandedDNA单链DNABridgeAmplificationSingle-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BridgeAmplificationSingle-strBridgeAmplificationDouble-strandedbridgeisformed延伸完成后形成双链DNA桥式结构桥式PCR扩增BridgeAmplificationDouble-strDenatureDouble-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DenatureDouble-StrandedBridgBridgeAmplificationSingle-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BridgeAmplificationSingle-strBridgeAmplificationBridgeamplificationcycleisrepeateduntilmultiplebridgesareformed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BridgeAmplificationBridgeampLinearizationdsDNAbridgesaredenatured双链DNA变性后，解开桥式结构，变成线性化的单链DNA线性化LinearizationdsDNAbridgesareReverseStrandCleavageReversestrandsarecleavedandwashedaway,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ReverseStrandCleavageReversBlockingFree3’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BlockingFree3’endsareblockRead1PrimerHybridizationSequencingprimer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Read1PrimerHybridizationSeqIlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析IlluminaSequencingWorkflow1LSequencingBySynthesis

Add4Fl-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）SequencingBySynthesis

Add4All4labelednucleotidesin1reaction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All4labelednucleotidesin1100MicronsClustersDNA簇100MicronsClustersDNA簇123789456TTTTTTT

G

T…T

G

C

T

A

C

G

A

T…Theidentityofeachbaseofaclusterisreadofffromsequentialimages根据每个点每轮反应读取的荧光信号序列，转换成相应的DNA序列照相读取序列123789456TTTTTTTGT…T25FourChannelSBSChemistry:GA,HiSeq,MiSeq

四通道SBS测序中收集对应波长通道的4张相片每个测序循环中，结合有不同碱基的DNA簇会在4张照片中的一张中出现每种DNA碱基具有独特的荧光波长（不同的颜色）FourChannelSBSChemistry:GA双通道SBS测序仅使用2张图像测序簇中掺入核苷酸T时仅在绿色通道中出现测序簇中掺入核苷酸C

时仅在红色通道中出现测序簇中掺入核苷酸A

时在绿色和红色通道中都出现测序簇中掺入核苷酸G

时在绿色和红色通道中都不出现将测序簇在两种通道中的荧光强度坐标以图表画出，即可相应地读出不同碱基TwoChannelSBS–NextSeq

双通道SBS双通道SBS测序仅使用2张图像TwoChannelSBSPairedEndSequencing双末端测序PairedEndSequencing双末端测序ThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisis

reallythebestwaytodo

sequencing(----100characters-------)Reference参考序列Single-reads单端序列信息…………Paired-reads双端序列信息序列更容易进行组装！！SequencingwithPairedEnds双末端测序ThisisreallythebestwaytoSequencedstrand测序生成链Blocked3’-ends被阻断的3’端Sequencedstrandisstrippedoff测序反应生成的片段被变性洗去3’-endsoftemplatestrandsandlawnprimersareunblocked与流动槽结合的DNA序列上3’端阻断被去除（同时也去除引物丛上的3’端阻断）PairedEndSequencing双末端测序Discard洗去丢弃SequencedstrandBlocked3’-endBridgeformation桥式结构3’extension3’延伸反应Single-strandedtemplateloopsovertoformabridgebyhybridizingwithalawnprimerDNA单链折叠后与其附近的引物丛杂交结合形成新的桥式结构3’-endsoflawnprimerisextended在DNA合成酶作用下，结合上模板的引物丛3’端开始延伸PairedEndSequencing双末端测序Bridgeformation3’extensionSiDoublestrandedDNA双链DNA(桥式结构)PairedEndSequencing双末端测序DoublestrandedDNAPairedEndOriginalforwardstrand原始的正链PairedEndSequencingBridgesarelinearizedandtheoriginalforwardtemplateiscleaved双链桥式结构被变性，形成线性结构的DNA簇；随后正义链被切除，留下只含有反义链的DNA簇双末端测序OriginalforwardstrandPairedReversestrandTemplate反义链模板Blocked3’-ends阻断3’端Sequencingprimer测序引物PairedEndSequencingFree3’endsofthereversetemplateandlawnprimersareblockedtopreventunwantedDNApriming为了防止测序过程中不需要的DNA延伸，对流动槽上结合的所有DNA分子的3’端进行封闭SequencingprimerishybridizedtoadaptersequenceRead2测序引物与待测DNA分子的接头序列杂交结合双末端测序ReversestrandBlocked3’-endsSSequencingBySynthesis2nd

Read

Add4Fl-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dNTP中的荧光标记和阻断基团被切除，可以继续下一轮反应X36-251Read2边合成边测序SequencingBySynthesis2ndReSequencingPairedEndLibrarieswithSingleIndexRead单标签（SingleIndex）双末端测序SequencingPairedEndLibrarieRead2SeqPrimer(HP7)Read1SeqPrimer(HP6)IndexSeqPrimer(HP8)123MultiplexSequencingUtilizes3SequencingReadsPairedEndTurnaround序列翻转SingleIndexSequencingUtilizes3

SequencingReads单index双末端测序使用3种测序引物，3次序列读取SequencingwithPairedEnds双末端测序（单标签）正向序列读取索引序列读取反向序列读取Read2SeqPrimerRead1SeqPrSequencingPairedEndLibrarieswithDualIndexRead双标签（DualIndex）双末端测序SequencingPairedEndLibrarie1234DualIndexSequencingUtilizes4SequencingReads双标签双末端测序包含4次测序读取，但是只用到3条不同测序引物（反向索引序列的读取使用了流动槽上锚定的P5序列）SequencingPairedEndLibrarieswithDualIndexRead双末端测序（双标签）适用于GAIIx,MiSeq,HiSeq1500/2000/2500正向序列读取正向索引序列读取反向索引序列读取反向序列读取仅进行化学合成，不照相PairedEndTurnaround序列翻转1234DualIndexSequencingUtilDualIndexSequencingUtilizes4SequencingReads双索引双末端测序包含4次测序读取，用到4条不同测序引物SequencingPairedEndLibrarieswithDualIndexRead双末端测序（双标签）适用于NextSeq,HiSeq3000/40001234正向序列读取正向索引序列读取反向索引序列读取反向序列读取PairedEndTurnaround序列翻转DualIndexSequencingUtilizesIlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析IlluminaSequencingWorkflow1LPrimaryAnalysis初级分析SecondaryAnalysis二级分析DataVisualization数据可视化DataAnalysisOverview数据分析总览PrimaryAnalysisSecondaryAnalAlignmentsandVariantDetection序列比对和突变检测Images/TIFFfiles图片文件BaseCalling读取碱基Intensities光强数据SoftwareOutputsPrimaryandSecondaryAnalysisOverviewAnalysisTypePrimaryAnalysis初级分析SecondaryAnalysis二级分析Sequencing测序ICS/RTAICS/RTAHiSeqAnalysisSoftware初级和二级数据分析总览AlignmentsandVariantDetectiSummary1LibraryPreparationcBotMninSeqMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiniSeqMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVALRMMSRBaseSpace总结文库制备簇生成测序数据分析Summary1LibraryPreparationcBo问题？

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ThankYou!ThankYou!46Illumina测序技术概述Illumina测序技术概述47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）的原理课程目标课程目标IlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析IlluminaSequencingWorkflow1LIlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析IlluminaSequencingWorkflow1L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②②②

Read1和Read2测序引物结合的区域③③

插入片段④④④

标签序列区域（Index）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IlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析IlluminaSequencingWorkflow1LWhatisaFlowCell?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WhatisaFlowCell?Eachlane簇生成SingleDNALibrary单个DNA文库分子AmplifiedClonalCluster扩增后的DNA克隆簇cBotSequencer簇生成SingleDNALibraryAmplifiedHybridizeFragment&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HybridizeFragment&ExtendAdaNewlysynthesizedstrand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NewlysynthesizedstrandOriginNOTE:SinglemoleculesbindtoflowcellinarandompatternNOTE:单个DNA分子以随机的方式与流动槽表面结合Single-StrandedDNA单链DNANOTE:Single-StrandedDNA单链DNABridgeAmplificationSingle-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BridgeAmplificationSingle-strBridgeAmplificationDouble-strandedbridgeisformed延伸完成后形成双链DNA桥式结构桥式PCR扩增BridgeAmplificationDouble-strDenatureDouble-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DenatureDouble-StrandedBridgBridgeAmplificationSingle-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BridgeAmplificationSingle-strBridgeAmplificationBridgeamplificationcycleisrepeateduntilmultiplebridgesareformed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BridgeAmplificationBridgeampLinearizationdsDNAbridgesaredenatured双链DNA变性后，解开桥式结构，变成线性化的单链DNA线性化LinearizationdsDNAbridgesareReverseStrandCleavageReversestrandsarecleavedandwashedaway,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ReverseStrandCleavageReversBlockingFree3’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BlockingFree3’endsareblockRead1PrimerHybridizationSequencingprimer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Read1PrimerHybridizationSeqIlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程文库制备簇生成测序数据分析IlluminaSequencingWorkflow1LSequencingBySynthesis

Add4Fl-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）SequencingBySynthesis

Add4All4labelednucleotidesin1reaction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All4labelednucleotidesin1100MicronsClustersDNA簇100MicronsClustersDNA簇123789456TTTTTTT

G

T…T

G

C

T

A

C

G

A

T…Theidentityofeachbaseofaclusterisreadofffromsequentialimages根据每个点每轮反应读取的荧光信号序列，转换成相应的DNA序列照相读取序列123789456TTTTTTTGT…T71FourChannelSBSChemistry:GA,HiSeq,MiSeq

四通道SBS测序中收集对应波长通道的4张相片每个测序循环中，结合有不同碱基的DNA簇会在4张照片中的一张中出现每种DNA碱基具有独特的荧光波长（不同的颜色）FourChannelSBSChemistry:GA双通道SBS测序仅使用2张图像测序簇中掺入核苷酸T时仅在绿色通道中出现测序簇中掺入核苷酸C

时仅在红色通道中出现测序簇中掺入核苷酸A

时在绿色和红色通道中都出现测序簇中掺入核苷酸G

时在绿色和红色通道中都不出现将测序簇在两种通道中的荧光强度坐标以图表画出，即可相应地读出不同碱基TwoChannelSBS–NextSeq

双通道SBS双通道SBS测序仅使用2张图像TwoChannelSBSPairedEndSequencing双末端测序PairedEndSequencing双末端测序ThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisis

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sequencing(----100characters-------)Reference参考序列Single-reads单端序列信息…………Paired-reads双端序列信息序列更容易进行组装！！SequencingwithPairedEnds双末端测序ThisisreallythebestwaytoSequencedstrand测序生成链Blocked3’-ends被阻断的3’端Sequencedstrandisstrippedoff测序反应生成的片段被变性洗去3’-endsoftemplatestrandsandlawnprimersareunblocked与流动槽结合的DNA序列上3’端阻断被去除（同时也去除引物丛上的3’端阻断）PairedEndSequencing双末端测序Discard洗去丢弃SequencedstrandBlocked3’-endBridgeformation桥式结构3’extension3’延伸反应Single-strandedtemplateloopsovertoformabridgebyhybridizingwithalawnprimerDNA单链折叠后与其附近的引物丛杂交结合形成新的桥式结构3’-endsoflawnprimerisextended在DNA合成酶作用下，结合上模板的引物丛3’端开始延伸PairedEndSequencing双末端测序Bridgeformation3’extensionSiDoublestrandedDNA双链DNA(桥式结构)PairedEndSequencing双末端测序DoublestrandedDNAPairedEndOriginalforwardstrand原始的正链PairedEndSequencingBridgesarelinearizedandtheoriginalforwardtemplateiscleaved双链桥式结构被变性，形成线性结构的DNA簇；随后正义链被切除，留下只含有反义链的DNA簇双末端测序OriginalforwardstrandPairedReversestrandTemplate反义链模板Blocked3’-ends阻断3’端Sequencingprimer测序引物PairedEndSequencingFree3’endsofthereversetemplateandlawnprimersareblockedtopreventunwantedDNApriming为了防止测序过程中不需要的DNA延伸，对流动槽上结合的所有DNA分子的3’端进行封闭SequencingprimerishybridizedtoadaptersequenceRead2测序引物与待测DNA分子的接头序列杂交结合双末端测序ReversestrandBlocked3’-endsSSequencingBySynthesis2nd

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Add4Fl-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dNTP中的荧光标记和阻断基团被切除，可以继续下一轮反应X36-251Read2边合成边测序SequencingBySynthesis2ndReSequencingPairedEndLibrarieswithSingleIndexRead单标签（SingleIndex）双末端测序SequencingPairedEndLibrarieRead2SeqPrimer(HP7)Read1SeqPrimer(HP6)IndexSe