Illumina測序介紹-課件

Illumina測序技術概述Illumina測序技術概述1課程結束時：清楚了解Illumina測序流程中的主要步驟能夠描述簇生成（ClusterGeneration）的過程

理解邊合成邊測序（SequencingbySynthesis）的原理課程目標課程目標IlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina測序流程文庫制備簇生成測序數(shù)據(jù)分析IlluminaSequencingWorkflow1LIlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina測序流程文庫制備簇生成測序數(shù)據(jù)分析IlluminaSequencingWorkflow1L文庫制備的目的是在需要測序的DNA片段兩端加上能夠與測序儀配合的接頭序列（Multiplexed,SR,PE）雙端標簽文庫文庫制備是決定測序?qū)嶒灣晒εc否的關鍵步驟①①①與流動槽（FlowCell）結合的區(qū)域②②②

Read1和Read2測序引物結合的區(qū)域③③

插入片段④④④

標簽序列區(qū)域（Index）文庫制備的目的是在需要測序的DNA片段兩端加上能夠與測序儀配IlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina測序流程文庫制備簇生成測序數(shù)據(jù)分析IlluminaSequencingWorkflow1LWhatisaFlowCell?Eachlaneisrandomlycoatedwithalawnofoligosthatarecomplementarytolibraryadapters每條通道中都隨機植入了能與文庫接頭互補結合的大量短DNA片段Clustergenerationoccursonaflowcell簇生成在流動槽（flowcell）上完成流動槽(FlowCell)是什么?Aflowcellisathickglassslidewithchannelsorlanes一種含有通道的厚玻璃片WhatisaFlowCell?Eachlane簇生成SingleDNALibrary單個DNA文庫分子AmplifiedClonalCluster擴增后的DNA克隆簇cBotSequencer簇生成SingleDNALibraryAmplifiedHybridizeFragment&ExtendAdaptersequence接頭序列3’extension3’延伸Surfaceofflowcellcoatedwithalawnofoligopairs流動槽表面隨機植入了大量引物鏈SingleDNAlibrariesarehybridizedtoprimerlawn變性后單鏈DNA文庫與流動槽上的引物雜交Boundlibrariesarethenextendedbypolymerases隨后在DNA合成酶的作用下，結合上的DNA文庫進行延伸片段雜交與延伸HybridizeFragment&ExtendAdaNewlysynthesizedstrand新合成鏈Originaltemplate原始模板鏈DenatureDouble-StrandedDNADiscard洗去丟棄Double-strandedmoleculeisdenatured雙鏈DNA分子被變性Originaltemplatewashedaway原始的模板鏈被洗去Newlysynthesizedstrandiscovalentlyattachedtoflowcellsurface新合成的DNA鏈以共價鍵連接的方式結合在流動槽表面雙鏈DNA變性NewlysynthesizedstrandOriginNOTE:SinglemoleculesbindtoflowcellinarandompatternNOTE:單個DNA分子以隨機的方式與流動槽表面結合Single-StrandedDNA單鏈DNANOTE:Single-StrandedDNA單鏈DNABridgeAmplificationSingle-strandedmoleculeflipsoverandformsabridgebyhybridizingtoadjacent,complementaryprimer共價鍵結合在流動槽表面的單鏈DNA分子與其附近的互補引物雜交，整條DNA分子折疊后形成一種類似于橋的結構。Hybridizedprimerisextendedbypolymerases在DNA合成酶作用下，雜交后的引物以單鏈DNA為模板進行延伸橋式PCR擴增3’extension3‘延伸反應BridgeAmplificationSingle-strBridgeAmplificationDouble-strandedbridgeisformed延伸完成后形成雙鏈DNA橋式結構橋式PCR擴增BridgeAmplificationDouble-strDenatureDouble-StrandedBridgeDouble-strandedbridgeisdenatured橋式結構的雙鏈DNA被變性Result:Twocopiesofcovalentlyboundsingle-strandedtemplates結果：形成兩條與流動槽表面共價鍵結合的DNA模板雙鏈DNA橋式結構變性DenatureDouble-StrandedBridgBridgeAmplificationSingle-strandedmoleculesflipovertohybridizetoadjacentprimers單鏈DNA分子再一次折疊后與附近的引物雜交結合Hybridizedprimerisextendedbypolymerase雜交后的引物再次在DNA合成酶的作用下延伸橋式PCR擴增BridgeAmplificationSingle-strBridgeAmplificationBridgeamplificationcycleisrepeateduntilmultiplebridgesareformed橋式擴增不斷重復發(fā)生直到形成數(shù)量足夠的DNA橋（與PCR反應類似，區(qū)別在于引物不是游離在溶液中，而是固定在流動槽表面）橋式PCR擴增BridgeAmplificationBridgeampLinearizationdsDNAbridgesaredenatured雙鏈DNA變性后，解開橋式結構，變成線性化的單鏈DNA線性化LinearizationdsDNAbridgesareReverseStrandCleavageReversestrandsarecleavedandwashedaway,leavingaclusterwithforwardstrandsonly與流動槽表面結合的DNA反鏈被切除并洗去，只留下正鏈，形成包含均一單鏈的DNA簇反鏈切除ReverseStrandCleavageReversBlockingFree3’endsareblockedtopreventunwantedDNApriming為了防止后續(xù)測序過程中不必要的DNA延伸，對流動槽上結合的所有DNA分子的3’端進行封閉DNA鏈封閉BlockingFree3’endsareblockRead1PrimerHybridizationSequencingprimer測序引物Sequencingprimerishybridizedtoadaptersequence將Read1測序引物加入流動槽，使其與待測DNA分子的接頭序列結合Read1引物雜交Read1PrimerHybridizationSeqIlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina測序流程文庫制備簇生成測序數(shù)據(jù)分析IlluminaSequencingWorkflow1LSequencingBySynthesis

Add4Fl-NTP’s+Polymerase加入4種不同熒光標記的dNTP和DNA合成酶IncorporatedFI-NTPimaged對結合上的熒光dNTP進行照相Terminator&fluorescentdyecleavedfromFI-NTP結合在DNA鏈上的熒光NTP中的熒光標記和阻斷基團被切除，可以繼續(xù)下一輪反應X36-251邊合成邊測序（SBS）SequencingBySynthesis

Add4All4labelednucleotidesin1reaction同時存在4種不同標記的核苷酸Higheraccuracy更高的準確性Noproblemswithhomopolymerrepeats不存在均聚物重復片段測序困難問題Incorporation結合Detection檢測Deblock去阻斷FluorRemoval

切除熒光基團NextCycle下一個循環(huán)ReversibleTerminatorChemistry可逆阻斷3‘阻斷基團切除位點熒光基團去阻斷后自由的3‘OH端All4labelednucleotidesin1100MicronsClustersDNA簇100MicronsClustersDNA簇123789456TTTTTTT

G

T…T

G

C

T

A

C

G

A

T…Theidentityofeachbaseofaclusterisreadofffromsequentialimages根據(jù)每個點每輪反應讀取的熒光信號序列，轉換成相應的DNA序列照相讀取序列123789456TTTTTTTGT…T25FourChannelSBSChemistry:GA,HiSeq,MiSeq

四通道SBS測序中收集對應波長通道的4張相片每個測序循環(huán)中，結合有不同堿基的DNA簇會在4張照片中的一張中出現(xiàn)每種DNA堿基具有獨特的熒光波長（不同的顏色）FourChannelSBSChemistry:GA雙通道SBS測序僅使用2張圖像測序簇中摻入核苷酸T時僅在綠色通道中出現(xiàn)測序簇中摻入核苷酸C

時僅在紅色通道中出現(xiàn)測序簇中摻入核苷酸A

時在綠色和紅色通道中都出現(xiàn)測序簇中摻入核苷酸G

時在綠色和紅色通道中都不出現(xiàn)將測序簇在兩種通道中的熒光強度坐標以圖表畫出，即可相應地讀出不同堿基TwoChannelSBS–NextSeq

雙通道SBS雙通道SBS測序僅使用2張圖像TwoChannelSBSPairedEndSequencing雙末端測序PairedEndSequencing雙末端測序ThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisisreallythebestwaytodosequencingThisis

reallythebestwaytodo

sequencing(----100characters-------)Reference參考序列Single-reads單端序列信息…………Paired-reads雙端序列信息序列更容易進行組裝??！SequencingwithPairedEnds雙末端測序ThisisreallythebestwaytoSequencedstrand測序生成鏈Blocked3’-ends被阻斷的3’端Sequencedstrandisstrippedoff測序反應生成的片段被變性洗去3’-endsoftemplatestrandsandlawnprimersareunblocked與流動槽結合的DNA序列上3’端阻斷被去除（同時也去除引物叢上的3’端阻斷）PairedEndSequencing雙末端測序Discard洗去丟棄SequencedstrandBlocked3’-endBridgeformation橋式結構3’extension3’延伸反應Single-strandedtemplateloopsovertoformabridgebyhybridizingwithalawnprimerDNA單鏈折疊后與其附近的引物叢雜交結合形成新的橋式結構3’-endsoflawnprimerisextended在DNA合成酶作用下，結合上模板的引物叢3’端開始延伸PairedEndSequencing雙末端測序Bridgeformation3’extensionSiDoublestrandedDNA雙鏈DNA(橋式結構)PairedEndSequencing雙末端測序DoublestrandedDNAPairedEndOriginalforwardstrand原始的正鏈PairedEndSequencingBridgesarelinearizedandtheoriginalforwardtemplateiscleaved雙鏈橋式結構被變性，形成線性結構的DNA簇；隨后正義鏈被切除，留下只含有反義鏈的DNA簇雙末端測序OriginalforwardstrandPairedReversestrandTemplate反義鏈模板Blocked3’-ends阻斷3’端Sequencingprimer測序引物PairedEndSequencingFree3’endsofthereversetemplateandlawnprimersareblockedtopreventunwantedDNApriming為了防止測序過程中不需要的DNA延伸，對流動槽上結合的所有DNA分子的3’端進行封閉SequencingprimerishybridizedtoadaptersequenceRead2測序引物與待測DNA分子的接頭序列雜交結合雙末端測序ReversestrandBlocked3’-endsSSequencingBySynthesis2nd

Read

Add4Fl-NTP’s+Polymerase加入4種不同熒光標記的dNTP和DNA合成酶IncorporatedFI-NTPimaged對結合上的熒光dNTP進行照相Terminator&fluorescentdyecleavedfromFI-NTP結合在DNA鏈上的熒光dNTP中的熒光標記和阻斷基團被切除，可以繼續(xù)下一輪反應X36-251Read2邊合成邊測序SequencingBySynthesis2ndReSequencingPairedEndLibrarieswithSingleIndexRead單標簽（SingleIndex）雙末端測序SequencingPairedEndLibrarieRead2SeqPrimer(HP7)Read1SeqPrimer(HP6)IndexSeqPrimer(HP8)123MultiplexSequencingUtilizes3SequencingReadsPairedEndTurnaround序列翻轉SingleIndexSequencingUtilizes3

SequencingReads單index雙末端測序使用3種測序引物，3次序列讀取SequencingwithPairedEnds雙末端測序（單標簽）正向序列讀取索引序列讀取反向序列讀取Read2SeqPrimerRead1SeqPrSequencingPairedEndLibrarieswithDualIndexRead雙標簽（DualIndex）雙末端測序SequencingPairedEndLibrarie1234DualIndexSequencingUtilizes4SequencingReads雙標簽雙末端測序包含4次測序讀取，但是只用到3條不同測序引物（反向索引序列的讀取使用了流動槽上錨定的P5序列）SequencingPairedEndLibrarieswithDualIndexRead雙末端測序（雙標簽）適用于GAIIx,MiSeq,HiSeq1500/2000/2500正向序列讀取正向索引序列讀取反向索引序列讀取反向序列讀取僅進行化學合成，不照相PairedEndTurnaround序列翻轉1234DualIndexSequencingUtilDualIndexSequencingUtilizes4SequencingReads雙索引雙末端測序包含4次測序讀取，用到4條不同測序引物SequencingPairedEndLibrarieswithDualIndexRead雙末端測序（雙標簽）適用于NextSeq,HiSeq3000/40001234正向序列讀取正向索引序列讀取反向索引序列讀取反向序列讀取PairedEndTurnaround序列翻轉DualIndexSequencingUtilizesIlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina測序流程文庫制備簇生成測序數(shù)據(jù)分析IlluminaSequencingWorkflow1LPrimaryAnalysis初級分析SecondaryAnalysis二級分析DataVisualization數(shù)據(jù)可視化DataAnalysisOverview數(shù)據(jù)分析總覽PrimaryAnalysisSecondaryAnalAlignmentsandVariantDetection序列比對和突變檢測Images/TIFFfiles圖片文件BaseCalling讀取堿基Intensities光強數(shù)據(jù)SoftwareOutputsPrimaryandSecondaryAnalysisOverviewAnalysisTypePrimaryAnalysis初級分析SecondaryAnalysis二級分析Sequencing測序ICS/RTAICS/RTAHiSeqAnalysisSoftware初級和二級數(shù)據(jù)分析總覽AlignmentsandVariantDetectiSummary1LibraryPreparationcBotMninSeqMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiniSeqMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVALRMMSRBaseSpace總結文庫制備簇生成測序數(shù)據(jù)分析Summary1LibraryPreparationcBo問題？

問題？

ThankYou!ThankYou!46Illumina測序技術概述Illumina測序技術概述47課程結束時：清楚了解Illumina測序流程中的主要步驟能夠描述簇生成（ClusterGeneration）的過程

理解邊合成邊測序（SequencingbySynthesis）的原理課程目標課程目標IlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina測序流程文庫制備簇生成測序數(shù)據(jù)分析IlluminaSequencingWorkflow1LIlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina測序流程文庫制備簇生成測序數(shù)據(jù)分析IlluminaSequencingWorkflow1L文庫制備的目的是在需要測序的DNA片段兩端加上能夠與測序儀配合的接頭序列（Multiplexed,SR,PE）雙端標簽文庫文庫制備是決定測序?qū)嶒灣晒εc否的關鍵步驟①①①與流動槽（FlowCell）結合的區(qū)域②②②

Read1和Read2測序引物結合的區(qū)域③③

插入片段④④④

標簽序列區(qū)域（Index）文庫制備的目的是在需要測序的DNA片段兩端加上能夠與測序儀配IlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina測序流程文庫制備簇生成測序數(shù)據(jù)分析IlluminaSequencingWorkflow1LWhatisaFlowCell?Eachlaneisrandomlycoatedwithalawnofoligosthatarecomplementarytolibraryadapters每條通道中都隨機植入了能與文庫接頭互補結合的大量短DNA片段Clustergenerationoccursonaflowcell簇生成在流動槽（flowcell）上完成流動槽(FlowCell)是什么?Aflowcellisathickglassslidewithchannelsorlanes一種含有通道的厚玻璃片WhatisaFlowCell?Eachlane簇生成SingleDNALibrary單個DNA文庫分子AmplifiedClonalCluster擴增后的DNA克隆簇cBotSequencer簇生成SingleDNALibraryAmplifiedHybridizeFragment&ExtendAdaptersequence接頭序列3’extension3’延伸Surfaceofflowcellcoatedwithalawnofoligopairs流動槽表面隨機植入了大量引物鏈SingleDNAlibrariesarehybridizedtoprimerlawn變性后單鏈DNA文庫與流動槽上的引物雜交Boundlibrariesarethenextendedbypolymerases隨后在DNA合成酶的作用下，結合上的DNA文庫進行延伸片段雜交與延伸HybridizeFragment&ExtendAdaNewlysynthesizedstrand新合成鏈Originaltemplate原始模板鏈DenatureDouble-StrandedDNADiscard洗去丟棄Double-strandedmoleculeisdenatured雙鏈DNA分子被變性Originaltemplatewashedaway原始的模板鏈被洗去Newlysynthesizedstrandiscovalentlyattachedtoflowcellsurface新合成的DNA鏈以共價鍵連接的方式結合在流動槽表面雙鏈DNA變性NewlysynthesizedstrandOriginNOTE:SinglemoleculesbindtoflowcellinarandompatternNOTE:單個DNA分子以隨機的方式與流動槽表面結合Single-StrandedDNA單鏈DNANOTE:Single-StrandedDNA單鏈DNABridgeAmplificationSingle-strandedmoleculeflipsoverandformsabridgebyhybridizingtoadjacent,complementaryprimer共價鍵結合在流動槽表面的單鏈DNA分子與其附近的互補引物雜交，整條DNA分子折疊后形成一種類似于橋的結構。Hybridizedprimerisextendedbypolymerases在DNA合成酶作用下，雜交后的引物以單鏈DNA為模板進行延伸橋式PCR擴增3’extension3‘延伸反應BridgeAmplificationSingle-strBridgeAmplificationDouble-strandedbridgeisformed延伸完成后形成雙鏈DNA橋式結構橋式PCR擴增BridgeAmplificationDouble-strDenatureDouble-StrandedBridgeDouble-strandedbridgeisdenatured橋式結構的雙鏈DNA被變性Result:Twocopiesofcovalentlyboundsingle-strandedtemplates結果：形成兩條與流動槽表面共價鍵結合的DNA模板雙鏈DNA橋式結構變性DenatureDouble-StrandedBridgBridgeAmplificationSingle-strandedmoleculesflipovertohybridizetoadjacentprimers單鏈DNA分子再一次折疊后與附近的引物雜交結合Hybridizedprimerisextendedbypolymerase雜交后的引物再次在DNA合成酶的作用下延伸橋式PCR擴增BridgeAmplificationSingle-strBridgeAmplificationBridgeamplificationcycleisrepeateduntilmultiplebridgesareformed橋式擴增不斷重復發(fā)生直到形成數(shù)量足夠的DNA橋（與PCR反應類似，區(qū)別在于引物不是游離在溶液中，而是固定在流動槽表面）橋式PCR擴增BridgeAmplificationBridgeampLinearizationdsDNAbridgesaredenatured雙鏈DNA變性后，解開橋式結構，變成線性化的單鏈DNA線性化LinearizationdsDNAbridgesareReverseStrandCleavageReversestrandsarecleavedandwashedaway,leavingaclusterwithforwardstrandsonly與流動槽表面結合的DNA反鏈被切除并洗去，只留下正鏈，形成包含均一單鏈的DNA簇反鏈切除ReverseStrandCleavageReversBlockingFree3’endsareblockedtopreventunwantedDNApriming為了防止后續(xù)測序過程中不必要的DNA延伸，對流動槽上結合的所有DNA分子的3’端進行封閉DNA鏈封閉BlockingFree3’endsareblockRead1PrimerHybridizationSequencingprimer測序引物Sequencingprimerishybridizedtoadaptersequence將Read1測序引物加入流動槽，使其與待測DNA分子的接頭序列結合Read1引物雜交Read1PrimerHybridizationSeqIlluminaSequencingWorkflow1LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid2ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq3Sequencing4DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina測序流程文庫制備簇生成測序數(shù)據(jù)分析IlluminaSequencingWorkflow1LSequencingBySynthesis

Add4Fl-NTP’s+Polymerase加入4種不同熒光標記的dNTP和DNA合成酶IncorporatedFI-NTPimaged對結合上的熒光dNTP進行照相Terminator&fluorescentdyecleavedfromFI-NTP結合在DNA鏈上的熒光NTP中的熒光標記和阻斷基團被切除，可以繼續(xù)下一輪反應X36-251邊合成邊測序（SBS）SequencingBySynthesis

Add4All4labelednucleotidesin1reaction同時存在4種不同標記的核苷酸Higheraccuracy更高的準確性Noproblemswithhomopolymerrepeats不存在均聚物重復片段測序困難問題Incorporation結合Detection檢測Deblock去阻斷FluorRemoval

切除熒光基團NextCycle下一個循環(huán)ReversibleTerminatorChemistry可逆阻斷3‘阻斷基團切除位點熒光基團去阻斷后自由的3‘OH端All4labelednucleotidesin1100MicronsClustersDNA簇100MicronsClustersDNA簇123789456TTTTTTT

G

T…T

G

C

T

A

C

G

A

T…Theidentityofeachbaseofaclusterisreadofffromsequentialimages根據(jù)每個點每輪反應讀取的熒光信號序列，轉換成相應的DNA序列照相讀取序列123789456TTTTTTTGT…T71FourChannelSBSChemistry:GA,HiSeq,MiSeq

四通道SBS測序中收集對應波長通道的4張相片每個測序循環(huán)中，結合有不同堿基的DNA簇會在4張照片中的一張中出現(xiàn)每種DNA堿基具有獨特的熒光波長（不同的顏色）FourChannelSBSChemistry:GA雙通道SBS測序僅使用2張圖像測序簇中摻入核苷酸T時僅在綠色通道中出現(xiàn)測序簇中摻入核苷酸C

時僅在紅色通道中出現(xiàn)測序簇中摻入核苷酸A

時在綠色和紅色通道中都出現(xiàn)測序簇中摻入核苷酸G

時在綠色和紅色通道中都不出現(xiàn)將測序簇在兩種通道中的熒光強度坐標以圖表畫出，即可相應地讀出不同堿基TwoChannelSBS–NextSeq

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sequencing(----100characters-------)Reference參考序列Single-reads單端序列信息…………Paired-reads雙端序列信息序列更容易進行組裝??！SequencingwithPairedEnds雙末端測序ThisisreallythebestwaytoSequencedstrand測序生成鏈Blocked3’-ends被阻斷的3’端Sequencedstrandisstrippedoff測序反應生成的片段被變性洗去3’-endsoftemplatestrandsandlawnprimersareunblocked與流動槽結合的DNA序列上3’端阻斷被去除（同時也去除引物叢上的3’端阻斷）PairedEndSequencing雙末端測序Discard洗去丟棄SequencedstrandBlocked3’-endBridgeformation橋式結構3’extension3’延伸反應Single-strandedtemplateloopsovertoformabridgebyhybridizingwithalawnprimerDNA單鏈折疊后與其附近的引物叢雜交結合形成新的橋式結構3’-endsoflawnprimerisextended在DNA合成酶作用下，結合上模板的引物叢3’端開始延伸PairedEndSequencing雙末端測序Bridgeformation3’extensionSiDoublestrandedDNA雙鏈DNA(橋式結構)PairedEndSequencing雙末端測序DoublestrandedDNAPairedEndOriginalforwardstrand原始的正鏈PairedEndSequencingBridgesarelinearizedandtheoriginalforwardtemplateiscleaved雙鏈橋式結構被變性，形成線性結構的DNA簇；隨后正義鏈被切除，留下只含有反義鏈的DNA簇雙末端測序OriginalforwardstrandPairedReversestrandTemplate反義鏈模板Blocked3’-ends阻斷3’端Sequencingprimer測序引物PairedEndSequencingFree3’endsofthereversetemplateandlawnprimersareblockedtopreventunwantedDNApriming為了防止測序過程中不需要的DNA延伸，對流動槽上結合的所有DNA分子的3’端進行封閉SequencingprimerishybridizedtoadaptersequenceRead2測序引物與待測DNA分子的接頭序列雜交結合雙末端測序ReversestrandBlocked3’-endsSSequencingBySynthesis2nd

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Add4Fl-NTP’s+Polymerase加入4種不同熒光標記的dNTP和DNA合成酶IncorporatedFI-NTPimaged對結合上的熒光dNTP進行照相Terminator&fluorescentdyecleavedfromFI-NTP結合在DNA鏈上的熒光dNTP中的熒光標記和阻斷基團被切除，可以繼續(xù)下一輪反應X36-251Read2邊合成邊測序SequencingBySynthesis2ndReSequencingPairedEndLibrarieswithSingleIndexRead單標簽（SingleIndex）雙末端測序SequencingPairedEndLibrarieRead2SeqPrimer(HP7)Read1SeqPrimer(HP6)IndexSe