Illumina测序原理.pptx

Illumina测序原理.pptx

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Illumina测序原理,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,4,3,测序工作流程概览,1,DNA（0.1-1.0ug）,Librarypreparation,Clustergeneration,1,2,Imageacquisition,Sequencing,测序过程概览SequencingOverview,制备文库LibraryPreparation,LibraryPrepOverview（gDNA）,DNATemplate1-5g，50lpurifiedDNAOD260/280=1.8-2.0AsintactaspossibleLibraryYield500-1000ngOD260/280=1.8SR:

150-250bpPE:

300-650bpMW=sizeinbpx650,DNA片段化（Fragmentation）,我们用的片段化方法CovarisBioruptor别的可选的片段化方法NebulizerSonicationHydroShearEnzymaticOthers,打碎,Covaris,修补末端,T4,Klenow,T4PNK,加A碱基,Klenowexo-,5,3,5,T,3,T,A,A,3,5,T,5,3,T,T,3,5,T,A,3,5,2%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&Excision,PCR扩增,5,T,3,A,5,A,T,3,富集,5,T,3,A,5,A,T,3,退火,5,T,3,A,5,A,T,3,3,A,5,T,3,T,A,5,延伸,5,3,5,T,3,T,A,A,10-12Cycles,LibraryQC,双侧测序:

500bp+100bp,单侧测序:

250bp+100bp,LibraryPreparationSummary,RepairEnds,AddanAtothe3Ends,FragmentDNA,SizeSelectonGel,LigateAdapters,PCR,Fragments800bp,BluntEndFragmentswith5Phosphorylatedends,3-dAOverhang,Adapter-ModifiedEnds,300-600bpFragments,AmplifiedDNAwithAdapters,GenomicDNALibrary,PurifiedGenomicDNA,QCLibrary,FASTLIBRARYPREPINLESSTHAN2HOURSClosedtubeDNAfragmentationTransposon-mediatedlibrarypreparationUltra-lowinputrequirements（50ng）ENABLESARANGEOFCEANDNGSAPPLICATIONS,EpicentreNexteraTechnologySingleTube,RapidLibraryPrep,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,OH,TemplateHybridization,BridgeAmplification,循环2528次,Lin_Blocking_PrimerHyb,边合成边测序SequencingbySynthesis,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）,SequencingbySynthesis,A,C,G,T,DataAnalysis,TTTTTTTGT,TGCTACGAT,SingleReadSequencingSummary,DataAnalysisImageAnalysisBaseCallingAssembly,ClusterGenerationTemplateHybBridgeAmplificationLin_Block_Hyb,SequencingIncorporationImage-takingCleaving,LibraryPreparationFrag_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SingleReadSequencing,Sequencereads,两侧测序Paired-EndSequencing,两种植有引物的测序芯片GraftedFlowCells:

SRvsPE,8oxoG-P7U-P5,OH,OH,U,8oxo-G,SingleReadPeriodateLinearization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,Read2:

Resynthesis&SBS,（Read1）读第一链（Read2）读Index（也称作Barcode，6bptag）（Read3）读第二链,1,2,3,Multiplexing,配对测序Mate-PairSequencing,2kb10kb,B,B,Paired-EndSequencing,数据分析DataAnalysis,数据分析流程DataAnalysisWorkflow,SYSTEMCONTROLSOFTWAREReadGeneration,CASAVAAlignments,variations,builds,VISUALIZATIONGenomeStudio,orfavoritebrowser,PrimaryAnalysisHCS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SecondaryAnalysisCASAVA片段组装，序列比较，SNP识别，indel识别，mRNA/miRNA定量VisualizationGenomeStudio数据的直观展示,IlluminaDataAnalysisTools,RTA=OLB,HCSRTA,basecallsqualityscores,readssummariesallelesSNPsindelscounts（RNA）,GenomeStudio,OLB,images,basecallsqualityscores,（optional）,CASAVA,CASAVA,+,HCSRTA,=,（SCS/RTA）+OLB,+,CASAVA,（resultsusingonlySCS/RTAandCASAVAarethesameasacomboofRTA,OLBandCASAVA）,CASAVA,分组：

Separatingthemultiplexedsamplesbeforealignment.组装：

Alignment（Gerald）byELANDv2Gappedalignments（upto20bases）.Multiseed（maximum4）.多态：

TheSNPcaller突变：

Theindelfinder（onlyonpairedendruns）Reportsinsertionsanddeletionsuptohalfthereadsize.定量：

mRNA-seqcounting,NGSSequencingData,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OneASCIIcharacterperbase,QualityScore,QV=-10xlgPe,