二代测序实验与测序原理.ppt

二代测序的建库与测序原理,何有裕yyheyyhe上海生物信息技术研究中心上海众信生物技术有限公司苏州众信生物技术有限公司,内容,样本处理与测序原理简介罗氏454Illuminasolexa原始数据质量控制,TruSeqRNAandDNASamplePreparation,ClusterGenerationOverview,1000-6000moleculespercluster,OH,ClusterGeneration,TemplateHybridization,diol,diol,1stcycledenaturation,ClusterGeneration,BridgePCR,Templatepreparation-bridgeRCR,Adaptorligation,Surfaceattachment,Bridgeamplification,Denaturation,TrendsinGenet24:133(2008),Firstbaseincorporated,Cycle1:Addsequencingreagents,DetectSignal,CleaveTerminatorandDye,Cycle2-n:Addsequencingreagentsandrepeat,SequencingbySynthesisOverview,Cyclicreversibletermination,AllfourlabeledreversibleterminatorsareaddedpercycleRemoveunincorporatedbasesanddetectsignalRemovetheterminatinggroupandthefluorescentdye,TrendsinGenet24:133(2008),Terminatinggroup,Fluorophorecleavage,NatRevGenet11:31(2010),Basecalling,FlowcelllayoutonGAII,Aflowcellcontains8lanes,Lane1,Lane2,Lane8,.,Column1Column2,Eachlanecontains2columns,Eachcolumncontains60tiles,Eachtileisimaged4timespercycle,PrimaryDataAnalysisByFirecrestandBustardinRTA/OLB,tiffimagefile,Intensityfile,Firecrest,Bustard,Sequencefile,OH,diol,diol,OH,ClusterGeneration,SequencingPrimerHybridization（Single测序方式处理步骤）,Sequencemultiplesamplesinthesamelanes,DNAinsert,Read1,IndexRead,Read2,DNAinsert,Index,IndexSP,Rd2SP,Rd1SP,Multiplexingmultiplesamplesinthesamelanes,Pair-end测序优势,Mate-pair建库和测序,MolecularEcologyResources(2011),Templatepreparation-emulsionPCR,TrendsinGenet24:133(2008),Pyrosequencing,SingledNTPtypeflowspercycleInorganicpyrophosphate(PPi)drivesvisiblelightthroughaseriesofreactionsRemoveunincorporatednucleotide,TrendsinGenet24:133(2008),Basecalling,Homopolymererror,GV6330,20,灵活的多样本标签技术,454、solexa测序模式,DetectH+releasedasavoltagechangefastCommonmicrochipdesignstandardslow-costmanufacturingSequencingvolumeisincreasing,Semiconductorsequencing,Fasta序列格式,Fastq文件用4行记录一条序列,第一行以字符开头，跟在后面的是序列标识和描述第二行是序列字符第三行以+字符开头，后面可以为空，或者和第一行一样第四行是第二行序列质量数据的编码，长度需和第二行一样,HWI-ST507:211:C18E6ACXX:2:1101:1688:19921:N:0:GAGTGGCGACAATTTTTTTTGATATTAATAAAGATAGAACTTTCTTCCTATGAGTTTTCTCTC+CCCFFDFFHHHHGJJGHIIJGIIJJJJIIJJHJJJJJIJJIIIGIIIJGGIHJDIJIGAHEHFFGHGHE,Example:,Illuminasequenceidentifiers,HWI-EAS364_0004:4:1:995:9044#0/1,Casava1.8以前的序列标识,Illuminasequenceidentifiers,HWI-ST507:211:C18E6ACXX:2:1101:1688:19921:N:0:GAGTGG,Casava1.8的序列标识,序列质量,附：Solexa1.3以前的quality计算公式是:,SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS.XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII.JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ.LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL.!#$%?ABCDEFGHIJKLMNOPQRSTUVWXYZ_abcdefghijklmnopqr|33596473100-00.9.403.9.401S-SangerPhred+33,rawreadstypically(0,40)X-SolexaSolexa+64,rawreadstypically(-5,40)I-Illumina1.3+Phred+64,rawreadstypically(0,40)J-Illumina1.5+Phred+64,rawreadstypically(3,40)with0=unused,1=unused,2=ReadSegmentQualityControlIndicator(bold)(Note:Seediscussionabove).L-Illumina1.8+Phred+33,rawreadstypically(0,41),Q值对应ASCII码,454原始数据图片、sff格式、fasta格式（qual）,HSAPGDX01D1KDAlength=181xy=1540_3788region=1run=R_2012_08_01_00_39_39ACGTGTTCTGAGCCATATTGCGGTACTGGAAGGTGCGCCTGCACTGTCTGAGCACTGGTCACTGCTCGATACCAATGAAGCCTTATTTGATGAGGCGCGCACCACGCAGGCGGCGACTATTATCTTCTCGTTTGATCCAGAATAACCAAATCGAAAACGCTGGCAAGGCACACAGGGGATAHSAPGDX01D1KDAlength=181xy=1540_3788region=1run=R_2012_08_01_00_39_3940404040404040393738363424231919192420191818262618181918202020252526192020222222252826242222222524282828292928303030262626272727313130282828303030302621212020262728242520202020191919272828303031302828303131323231313030303127242422202020222626222223161616192216131313162223232326262424261313111112121922181811111313182424242426262627292931333231312727272929282622,454原始数据长度分布（质控后一样）,Yield,datasizeproducedbysequencer.Reads,sequencedfragments.Readlengthandquality.Coveragefold,numberoftimesanucleotideisrepresented.Depth,theaveragecoveragefold.Coveragerate,ratiooftheregionsequencedtothewholegenome.Homopolymer,e.g.AAAAA,KeylabofsystemsbiologySIBS,ChineseAcademyofSciences,一些测序中提到的基本概念,通常深度测序数据处理流程,KeylabofsystemsbiologySIBS,ChineseAcademyofSciences,序列质量评估,FastQC:AqualitycontroltoolforhighthroughputsequencedataJavahttp:/www.bioinformatics.bbsrc.ac.uk/projects/fastqc/Function:,QCpipeline,原始数据的质控过滤,SequencelevelShortsequencesAdaptor/primerpolyA|TregionOveralllow-complexitysequence(Dust)Contamination/unwantedsequencesNs(lowqualityends)QualitylevelLowqualitybaseorregion目标：所有保留的都是高质量的，真正参与生物信息分析的数据。,Cleanreads,去掉含有接头序列的reads；当单端测