Illumina-测序原理

IlluminaDNA测序原理DavidChen北京源泉宜科生物科技有限公司目录概览制备文库(LibraryPreparation)在测序芯片上生成DNA簇(ClusterGeneration)边合成边测序(SequencingbySynthesis)双侧测序，配对测序(Paired-End,MatePair&Multiplexing)数据分析(DataAnalysis)LibraryPreparation制备文库Sequencing测序DataAnalysis数据分析ClusterGeneration生成DNA簇测序工作流程概览DNA(0.1-1.0ug)LibrarypreparationClustergeneration5’5’3’GTCAGTCAGTCACAGTCATCACCTAGCGTAGT123789456ImageacquisitionSequencing测序过程概览SequencingOverviewBasecallingTGCTACGAT…制备文库LibraryPreparationDNA片段化(Fragmentation)我们用的片段化方法–Covaris–Bioruptor别的可选的片段化方法–Nebulizer–Sonication–HydroShear®–Enzymatic–Others打碎Covaris修补末端T4,Klenow,T4PNK加A碱基AAAAAAKlenowexo-PPAA5’5’5’3’5’T3’TAAP3’5’T5’3’TT3’5’TA3’5’2%AgaroseGel电泳切胶选取DNA，对于单侧测序选取150-250bp，对于双侧测序选取600bp磁珠或Qiagen等回收试剂盒回收DNA片段AdaptorsLigation&ExcisionPCR扩增5’T3’A5’AT3’富集5’T3’A5’AT3’退火5’T3’A5’AT3’3’A5’T3’TA5’延伸5’3’5’T3’TAA10-12CyclesLibraryQC双侧测序:500bp+100bp单侧测序:250bp+100bpLibraryPreparationSummaryRepairEndsAddanAtothe3’EndsFragmentDNASizeSelectonGelLigateAdaptersPCRFragments800bpBluntEndFragmentswith5’–Phosphorylatedends3’-dAOverhangAdapter-ModifiedEnds300-600bpFragmentsAmplifiedDNAwithAdaptersGenomicDNALibraryPurifiedGenomicDNAQCLibrary生成DNA簇ClusterGeneration测序芯片(Flowcell)生成DNA簇(ClusterGeneration)每个簇有~1000-6000个DNA分子OHOHGraftedflowcell种有引物的芯片表面diolP7P5TemplateHybridizationdioldiolTemplateHybridization模板杂交dioldiolInitialextension启始延伸dioldiolDenaturation解链1stcycleannealing第一次退火dioldioln=25/28total1stcycledenaturation第一次变性dioldiol1stcycleextension第一次延伸dioldioldioldiol2ndcycledenaturation第二次变性2ndcycleannealing第二次退炎dioldioldiolBridgeAmplificationdioldioldiol2ndcycleextension第二次延伸循环25~28次Lin_Blocking_PrimerHybClusterAmplification扩增生成DNA簇OHdioldiolOHLinearization单链化OHBlockingwithddNTP()堵上3‘端DenatureandHybridization变性再杂交OH边合成边测序SequencingbySynthesisCAGTCATCACCTAGCGTA5’GTCAGTCAGTCAGT3’5’第一个碱基结合(Firstbaseincorporated)Cycle1:检测光信息(DetectSignal)切掉终止子和染料(CleaveTerminatorandDye)Cycle2-n:循环(Repeat)SequencingbySynthesisACGTDataAnalysis123789456TTTTTTTGT…TGCTACGAT…SingleReadSequencingSummaryDataAnalysisImageAnalysisBaseCallingAssemblyClusterGenerationTemplateHybBridgeAmplificationLin_Block_HybSequencingIncorporationImage-takingCleavingLibraryPreparationFrag_Repair_AddAAdaptorLig_ExcPCR_QC单侧测序、双侧测序、配对测序、多重测序SR,PE,MP,Multiplexing单侧测序SingleReadSequencingSequencereadsKnownsequenceNewsequence80-90%两侧测序Paired-EndSequencingSequencereadsKnownsequenceNewsequence95to99%OHOHdiolP7P5两种植有引物的测序芯片GraftedFlowCells:SRvsPE8oxoG-P7U-P5OHOHU8oxo-GSingleReadPeriodateLinearization单侧测序芯片一种引物有修饰PairedEndUracilSpecificExcisionReagent(USER)formamidopyrimidineglycosylase(fpg)双侧测序芯片两种引物都有修饰DenaturationandHybridization变性和杂交DenaturationandDe-Phosphorylation(PNK)变性，去掉磷酸基，活化另一侧引物OHOHResynthesisofP5Strand再次合成第二链OHP7Linearization(fpg)打断第一链OHBlockwithddNTPs堵上3’端DenaturationandHybridization变性再杂交Read2:Resynthesis&SBSSequencing第一链测序Read1Sequencing第二链侧序Read2(Read1)读第一链(Read2)读Index(也称作Barcode，6bptag)(Read3)读第二链123Multiplexing配对测序Mate-PairSequencing2kb–10kbBBBBBPaired-EndSequencing数据分析DataAnalysis数据分析流程DataAnalysisWorkflowSYSTEMCONTROLSOFTWAREReadGenerationCASAVAAlignments,variations,buildsVISUALIZATIONGenomeStudio,orfavoritebrowserPrimaryAnalysis–HCS仪器运转；收集信号–RTA实时图片分析；碱基识别；质量评估–OLB离线图片分析SecondaryAnalysis–CASAVA片段组装，序列比较，SNP识别，indel识别，mRNA/miRNA定量Visualization–GenomeStudio数据的直观展示IlluminaDataAnalysisToolsNGSSequencingDataASCIICharacterQ-scorePF(0,1)SequenceInstrumentRunIDLaneTileX-coordY-coordIndex#Read#数据质量值Qualityvalue数据质量值用ASCII码来记录QualityscorearerepresentedasASCIIcharacters(tosavespace)–一个ASCII码对应一个碱基OneASCIIcharacterperbaseQualityScore替代字母CharacterASCII码质量分值^9430_9531‘9632a9733b9834c9935d10036e10137f10238g10339QV=-10xlgPeThanks!