Illumina测序介绍_精品文档.pptx

Illumina测序介绍_精品文档.pptx

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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）的原理课程目程目标3Part#15045845_Rev.CFORRESEARCHUSEONLYIlluminaSequencingWorkflow11LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid22ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq33Sequencing44DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程序流程文文库制制备簇生成簇生成测序序数据分析数据分析4Part#15045845_Rev.CFORRESEARCHUSEONLYIlluminaSequencingWorkflow11LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid22ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq33Sequencing44DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程序流程文文库制制备簇生成簇生成测序序数据分析数据分析5Part#15045845_Rev.CFORRESEARCHUSEONLY文文库库制制备备的目的是在需要的目的是在需要测测序的序的DNADNA片段两端加上能片段两端加上能够够与与测测序序仪仪配合的接配合的接头头序列（序列（Multiplexed,SR,PEMultiplexed,SR,PE）双端标签文库文文库制制备是决定是决定测序序实验成功与否的成功与否的关关键步步骤与流与流动槽（槽（FlowCell）结合的区域合的区域Read1和和Read2测序引物序引物结合的区合的区域域插入片段插入片段标签序列区域（序列区域（Index）6Part#15045845_Rev.CFORRESEARCHUSEONLYIlluminaSequencingWorkflow11LibraryPreparationcBotMiSeqNextSeqHiSeq2500-Rapid22ClusterGenerationHiSeqHiScanSQGAIIxMiSeqNextSeq33Sequencing44DataAnalysisICS/RTACASAVAMSRBaseSpaceIllumina测序流程序流程文文库制制备簇生成簇生成测序序数据分析数据分析7Part#15045845_Rev.CFORRESEARCHUSEONLYWhatisaFlowCell?

EachlaneisrandomlycoatedwithEachlaneisrandomlycoatedwithalawnofalawnofoligosthatareoligosthatarecomplementarytolibraryadapterscomplementarytolibraryadapters每每条通道中都随机植入了能与文条通道中都随机植入了能与文库库接接头头互互补结补结合的大量短合的大量短DNADNA片段片段ClustergenerationoccursonaClustergenerationoccursonaflowcellflowcell簇生成在流簇生成在流动动槽（槽（flowcellflowcell）上完成）上完成流流动槽槽（FlowCell）是什么是什么?

AflowcellisathickglassslideAflowcellisathickglassslidewithchannelsorlaneswithchannelsorlanes一种含一种含有通道的厚有通道的厚玻玻璃片璃片8Part#15045845_Rev.CFORRESEARCHUSEONLY簇生成簇生成SingleDNALibrary单个个DNA文文库分子分子AmplifiedClonalCluster扩增后的增后的DNA克隆簇克隆簇cBotcBotSequencerSequencer9Part#15045845_Rev.CFORRESEARCHUSEONLYHybridizeFragment&ExtendAdaptersequence接接头序列序列3extension3延延伸伸Surfaceofflowcellcoatedwithalawnofoligopairs流流动槽表面随机植入了大量槽表面随机植入了大量引物引物链SingleDNAlibrariesareSingleDNAlibrariesarehybridizedtoprimerlawnhybridizedtoprimerlawn变变性后性后单链单链DNADNA文文库库与与流流动动槽上的引物槽上的引物杂杂交交BoundlibrariesarethenBoundlibrariesarethenextendedbypolymerasesextendedbypolymerases随后在随后在DNADNA合成合成酶酶的作的作用用下，下，结结合上的合上的DNADNA文文库进库进行延伸行延伸片段片段杂交交与与延伸延伸10Part#15045845_Rev.CFORRESEARCHUSEONLYNewlysynthesizedstrand新新合合成成链Originaltemplate原原始模板始模板链DenatureDouble-StrandedDNADiscard洗去洗去丢弃弃Double-strandedDouble-strandedmoleculeisdenaturedmoleculeisdenatured双双链链DNADNA分子被分子被变变性性OriginaltemplatewashedOriginaltemplatewashedawayaway原原始的模板始的模板链链被洗去被洗去NewlysynthesizedstrandNewlysynthesizedstrandiscovalentlyattachedtoiscovalentlyattachedtoflowcellsurfaceflowcellsurface新新合合成的成的DNADNA链链以共价以共价键键连连接的方式接的方式结结合在流合在流动动槽槽表面表面双双链DNA变性性11Part#15045845_Rev.CFORRESEARCHUSEONLYNOTE:

NOTE:

SingleSinglemoleculesmoleculesbindbindtoflowcellinatoflowcellinarandomrandompatternpatternNOTE:

NOTE:

单单个个DNADNA分子以随机分子以随机的方式与流的方式与流动动槽表面槽表面结结合合Single-StrandedDNA单链DNA12Part#15045845_Rev.CFORRESEARCHUSEONLYBridgeAmplificationSingle-strandedmoleculeflipsoverSingle-strandedmoleculeflipsoverandformsabridgebyhybridizingtoandformsabridgebyhybridizingtoadjacent,complementaryprimeradjacent,complementaryprimer共价共价键结键结合在流合在流动动槽表面的槽表面的单链单链DNADNA分子与其附近的互分子与其附近的互补补引物引物杂杂交，整条交，整条DNADNA分子折叠后形成一种分子折叠后形成一种类类似于似于桥桥的的结结构。

构。

HybridizedprimerisHybridizedprimerisextendedbypolymerasesextendedbypolymerases在在DNADNA合成合成酶酶作作用下，用下，杂杂交后的引物以交后的引物以单链单链DNADNA为为模板模板进进行延伸行延伸桥式式PCR扩增增3extension3延伸反延伸反应13Part#15045845_Rev.CFORRESEARCHUSEONLYBridgeAmplificationDouble-strandedbridgeisformedDouble-strandedbridgeisformed延延伸完成后形成双伸完成后形成双链链DNADNA桥桥式式结结构构桥式式PCR扩增增14Part#15045845_Rev.CFORRESEARCHUSEONLYDenatureDouble-StrandedBridgeDouble-strandedbridgeisDouble-strandedbridgeisdenatureddenatured桥桥式式结结构的双构的双链链DNADNA被被变变性性Result:

Result:

TwocopiesofcovalentlyboundTwocopiesofcovalentlyboundsingle-strandedtemplatessingle-strandedtemplates结结果：

形成两条与流果：

形成两条与流动动槽表面共槽表面共价价键结键结合的合的DNADNA模板模板双双链DNA桥式式结构构变性性15Part#15045845_Rev.CFORRESEARCHUSEONLYBridgeAmplificationSingle-strandedmoleculesflipoverSingle-strandedmoleculesflipovertohybridizetoadjacentprimerstohybridizetoadjacentprimers单单链链DNADNA分子再一次折叠后与附近分子再一次折叠后与附近的引物的引物杂杂交交结结合合HybridizedprimerisHybridizedprimerisextendedbypolymeraseextendedbypolymerase杂杂交交后的引物再次在后的引物再次在DNADNA合成合成酶酶的作用下延伸的作用下延伸桥式式PCR扩增增16Part#15045845_Rev.CFORRESEARCHUSEONLYBridgeAmplificationBridgeamplificationcyc