Day1_01_TS_Illumina_SBS二代测序简介-中国区武汉培训

©2011Illumina,Inc.Allrightsreserved.Illumina,illuminaDx,BeadArray,BeadXpress,cBot,CSPro,DASL,Eco,GeneticEnergy,GAIIx,GenomeAnalyzer,GenomeStudio,GoldenGate,HiScan,HiSeq,Infinium,iSelect,MiSeq,Nextera,Sentrix,Solexa,TruSeq,VeraCode,thepumpkinorangecolor,andtheGeneticEnergystreamingbasesdesignaretrademarksorregisteredtrademarksofIllumina,Inc.Allotherbrandsandnamescontainedhereinarethepropertyoftheirrespectiveowners.IIIumina二代测序技术简介童磊，PH.D.2012年7月12日2测序流程样本制备SamplePreparation初级分析PrimaryAnalysis簇生成ClusterGeneration测序Sequencing3dsNucleicAcid样本制备Samplepreparation123789456SequencingcyclesBasecallingTGCTACGAT…Illumina测序技术Singlemoleculearray簇生成Clustergrowth测序Sequencing5’5’3’CTGAACGATCGTCTGAACGATCGTTCTGAAT4测序流程:样本制备样本制备SamplePreparationPrimaryAnalysisClusterGenerationSequencing5gDNAmRNAChIPDNA文库制备DNA片段的两端加上测序接头文库制备smallRNAOtherApps6TruSeqDNA文库制备：叉状接头连接反应+PPAA5’5’T4DNALigase特征:一种接头室温进行反应·，以维持互补部分序列相互结合的状态P3’5’TTTAA3’5’3’5’Rd1SP5’P5Rd2SPIndexP75’7PPAA5’5’TTAA胶纯化2%AgaroseGelExciseDNAwithinthe~300bprangeP3’5’TTTTA3’5’3’5’3’5’3’5’3’5’8PCR富集(可选)PCRPCRPCR富集的步骤可以增加两端有接头的片段比例。同时把两端的互补序列不齐TTAA3’5’3’5’TA3’5’9文库（Library）–DNA插入片段加上两端的接头读长1测序引物（Read1SequencingPrimer）(Rd1SP)读长2测序引物（Read2SequencingPrimer）(Rd2SP)样本制备:Illumina文库结构解析DNAInsertAdapterAdapterIndexRd1SP5’P5Rd2SPIndexP75’10样本制备试剂盒KitDetailsIlluminaNexteraDNA96indexes快速，简洁TruSeqDNADNA24indexes低成本TruSeqRNAmRNA24indexesTruSeqSmallRNA48indexesTruSeqExome6indexes/富集TruSeqCustomEnrichment12indexes/富集11测序流程:DNA簇生成（ClusterGeneration）SamplePreparationPrimaryAnalysisDNA簇生成ClusterGenerationSequencing12Flowcell的表面覆盖了核酸丛。核酸丛的序列与核酸文库的接头互补。测序试剂流过核酸丛的同时发生测序反应。FlowCell介绍14簇生成的过程文库固定到FlowCell表面单链文库互补结合到flowcell表面的核酸链单链被复制复制链就通过核酸引物被固定在flowcell表面15复制链通过桥状结构与附近的核酸链互补结合以附近的核酸链为引物进行再次复制生成两条固定于flowcell表面的单链簇生成的过程文库扩增16桥式扩增反复进行，每条单链被复制为1000多个拷贝簇生成过程文库扩增17确定簇内每条链都是一致的(去掉互补链)簇生成过程EnsuringClusterHomogeneity(线性化处理)18簇内每一条链都进行末端封闭簇生成过程SealingtheStrands(封闭)19测序引物结合到簇内的DNA链。我们在这一步就拿到了成千上万的DNA簇。每个簇都是一个单一的文库序列。簇生成过程Read1测序引物杂交20测序流程:测序SamplePreparationPrimaryAnalysisClusterGeneration测序Sequencing21Illumina测序仪器HiScanSQGAIIxMiSeqHiSeq1000HiSeq2000数据量600GB1.5GB22FlowCell表面DNA簇Illumina边合成边测序（SequencingBySynthesis）5’3’单分子退火结合Read1引物23Illumina边合成边测序（SequencingBySynthesis）:成像5’5’3’单分子激光激发荧光放射1循环PoolofdNTPsCTGAACGATCGTT24Illumina边合成边测序（SequencingBySynthesis）:成像5’5’3’单分子激光激发荧光放射1循环PoolofdNTPsCTAACGATCGTTG225Illumina边合成边测序（SequencingBySynthesis）:成像5’5’3’单分子激光激发荧光放射1循环PoolofdNTPsCTAAGATCGTTG23C265’5’3’SingleMoleculeView激光激发荧光放射1CyclesPoolofdNTPsCTAAGATCGTTG23C4Illumina边合成边测序（SequencingBySynthesis）:成像27Illumina边合成边测序（SequencingBySynthesis）：初级序列分析SamplePreparation初级序列分析PrimaryAnalysisClusterGenerationSequencing28从图片中提取每个cycle的信息首先鉴别，定位，清点DNA簇初级分析循环1-4=28DNA簇29质量参数（Qualityscores-QScores)分配给所有的碱基分析Qscores反映了碱基分析出错的可能性初级分析QScoreProbabilityoferrorQ301in1000Q201in100Cycles1-4=TGCT30关键概念31ClustersonFlowCellSurface什么是双末端测序-Paired-End(PE)Sequencing?5’Read1sequence(100bp)CTAAGATCTTGCG5’3’SingleMoleculeViewRead1sequence32双末端测序:DNA簇重长SinglemoleculearrayClustergrowth33FlowCell表面的DNA簇双末端测序:从反向端开始测序CTAAGATCTTGCG5’5’3’单分子Read2引物结合34双末端测序:两端的特异序列singlereadpaired-end100bp100bp100bp35为什么多样本测序-Multiplex?HiSeq2000–1Flowcell=300Gb1lane=38Gbofsequence1Lane=180Million特异序列(簇)举例:典型的基因表达分析，每个样本需要500-1000万序列（DNA簇）每条通道18个样本每个Flowcell144个样本36多样本测序通过6bp的索引序列,我们每条lane可以同时进行24个样本的测序数据分析的时候根据索引序列自动分辨样本.37Read1多样本测序38HowMultiplexingWorksRead1IndexRead多样本测序39Read1IndexReadRead2多样本测序40dsNucleicAcid样本制备Samplepreparation123789456SequencingcyclesBasecallingTGCTACGAT…Illumina测序Singlemoleculearray簇生成Clustergrowth测序Sequencing5’5’3’CTGAACGATCGTCTGAACGATCGTTCTGAAT41培训录像:–TruSeq:DNASamplePreparation–Sequencing:IlluminaTechnology–Sequencing:PrimaryAnalysisV1.9文件:–IlluminaAdapterandIndexSequences–TruSeqSamplePrepKitsDataSheet文献:–Bentleyetal.(2008)Accuratewholehumangenomesequencingusingreversibleterminatorchemistry.Nature456:53-59其他信息：©2011Illumina,Inc.Allrightsreserved.Illumina,illuminaDx,BeadArray,BeadXpress,cBot,CSPro,DASL,Eco,GeneticEnergy,GAIIx,GenomeAnalyzer,GenomeStudio,GoldenGate,HiScan,HiSeq,Infinium,iSelect,MiSeq,Nextera,Sentrix,Solexa,TruSeq,VeraCode,thepumpkinorangecolor,andtheGeneticEnergystreamingbasesdesignaretrademarksorregisteredtrademarksofIllumina,Inc.Allotherbrandsandnamescontainedhereinarethepropertyoftheirrespectiveowners.Questions?THANKYOUFORATTENDING