BSA-seq

Lecture4：GeneticMappingUsingthe2ndGenerationSequencingMethod2016年3月25日Mappingwhengenome/linkagemapavailableWhenGeneticMapAvailable•Chooseamarkerfromalinkagegroup–Linkageanalysis•Chooseanothermarker~20cMapart–Linkageanalysis•……•Chooseamarkerfromanotherlinkagegroup,untilthetraitislinkedwithamarker!Finemappingwhengenome/linkagemapavailableTraitM1M2MM1M2M3M4M5TraitIncreasepopulationsize,morerecombinantBulkSegregationAnalysis(BSA)•Genotyping100individualsusing100markers•Poolof10individualswithonephenotype,10individualswithanotherphenotype•Genotypingthetwopools•Whynotpooling50individuals?Exam1BSAdueonApril81.WhenBSAisappliedtogeneticanalysisofaqualitativetraitusingSSR(orothertraditionalmarkers),differentnumberofindividualsarepooledforeach(extreme)phenotype.Somescientistspool5individualstogether,whileothersmaypool10,or20.Pleaseexplainsthemeritsanddrawbackstopoollargenumberorlownumberofindividuals.2.WhatifBSA+DNA-seqratherthanSSRisusedforabovecase?Canwepool1000individualsandusethesequencingdataforfinemapping?3.Forfinemapping,alargepopulationisrequired.Shouldwegrowallindividualsorrecombinantsonlytillflowering?Why?4.CanweuseBSAmethodforfinemapping?Whyorwhynot?Sequencing•Sangersequencing•Next(2nd)generationsequencing–454pyrosequencing–Illumina(Solexa)sequencing–SOLiDsequencing–IonTorrentsemiconductorsequencing•3rdgenerationsequencing:noPCRrequired,longreads,fast–NanoporeDNAsequencing–TunnellingcurrentsDNAsequencing–Sequencingbyhybridization–Sequencingwithmassspectrometry–MicrofluidicSangersequencing–Microscopy-basedtechniques–RNAPsequencing•4thgenerationsequencing:–Whatdoyouexpect?Howcanweusethe2ndgenerationsequencingtechniquetomapagene(s)controllingthetraitofinterest?2ndGenerationSequencing:•Randomgenomicsequences(shearedlibrary)•Largedata•CheapCompleteSequencingof100BC1Individuals……P1P2P1P2-+--+++--+--+++-+++--+……---++-++-+---+----Re-sequencing100BC1Individuals……P1P2P1P2-+--+++--+--+++-+++--+……---++-++-+---+----PredictingHaplotype……P1P2P1P2-+--+++--+--+++-+++--+……---++-++-+---+----Predictinggenotypesofallindividuals……P1P2YFGP1P2-+--+++--+--+++-+++--+……---++-++-+---+----RestrictionsiteassociatedDNA(RAD)markers•RADmarkerscanbe•Dominant:Presenceorabsenceoftherestrictionlocus•Codominant:SNPsofthesequencesatthesamelocusRADGenotyping100BC1IndividualsP1P2P1P2-+--+++--+--+++-+++--+……---++-++-+---+----PoolingExtremePenotypes-++++++++++++++++++……--------------------……P1P2P1P2PhenotypeMapthegenecontrollingtheredflower(method1:Genomicseqofextremepools)•ABC1population,with50individualswithRedflower,50withWhiteflower(RisdominantoverW)•Poolthetissuesof50individualswithR,extractgenomicDNA•Poolthetissuesof50individualswithW,extractgenomicDNA•Sequencethetwopools(20X)•SearchforregionsthatarehomozygousintheWpoolbutheterozygousintheRpoolCTCTCTCTCTCTCTTTTTTTTTRedPoolWhitepoolCTGTGCAGTCGGTAAGTACCGTACCAAGTGCAGCCTGTGCAGTCGGTAAGTACCGTATCAAGTGCAGCRedflowergeneWhiteflowergeneMapthegenecontrollingtheredflower(method2:RNA-seqofextremepools)•ABC1population,with50individualswithRedflower,50withWhiteflower(RisdominantoverW)•Poolthetissuesof50individualswithR,extracttotalRNA•Poolthetissuesof50individualswithW,extractTotalRNA•Sequencethetwopools(5Gforeach)•SearchforregionsthatarehomozygousintheWpoolbutheterozygousintheRpoolCTCTCTCTCTCTCTTTTTTTTTRedPoolWhitepoolCTGTGCAGTCGGTAAGTACCGTACCAAGTGCAGCCTGTGCAGTCGGTAAGTACCGTATCAAGTGCAGCRedflowergeneWhiteflowergeneMapthegenecontrollingtheredflower•AF2population,with50individualswithRedflower,50withWhiteflower(RisdominantoverW)•Poolthetissuesof50individualswithR,extracttotalRNA•Poolthetissuesof50individualswithW,extractTotalRNA•Sequencethetwopools(5Gforeach)•SearchforregionsthatarehomozygousintheWpoolbutheterozygousintheRpoolCCCTCCCCCTCTCCTTTTTTTTRedPoolWhitepoolCTGTGCAGTCGGTAAGTACCGTACCAAGTGCAGCCTGTGCAGTCGGTAAGTACCGTATCAAGTGCAGCRedflowergeneWhiteflowergeneMapthegenecontrollingtheredflower•AF2population,with50individualswithRedflower,50withWhiteflower(RissemidominantoverW;heterozygotesarepink)•Poolthetissuesof50individualswithR,extracttotalRNA•Poolthetissuesof50individualswithW,extractTotalRNA•Sequencethetwopools(5Gforeach)•SearchforregionsthatarehomozygousintheWpoolbutheterozygousintheRpoolCCCCCCCCCCCCCCTTTTTTTTRedPoolWhitepoolCTGTGCAGTCGGTAAGTACCGTACCAAGTGCAGCCTGTGCAGTCGGTAAGTACCGTATCAAGTGCAGCRedflowergeneWhiteflowergene×SegregatingpopulationP1P2pool1RNApool2RNAIlluminaRNAseqFastqfilesmaptogenome(tophat)BamalignmentfilesSNPfilescallingSNP(samtools)CalculateSNP-indexbetweentwobulksIdentifylinkedregionsVerificationusingmarkersFlowchartforSequenceAnalysisofBSA+RNA-seq1.Fastq文件测序公司给的fastq文件分为两类：rawfastq和cleanfastq。Rawfastq为原始的下机文件，而cleanfastq为过滤掉低质量reads的文件；一般mapping只需要cleanfastq文件就足够了。2.将fastq文件map到参考基因组，输出文件为bam格式的二进制比对文件脚本1star_map_samtools_index.pl调用了STAR、picard以及samtools等软件，将fastq文件map到参考基因组。1star_map_samtools_index.pl需要三个参数：dir.list文件为fastq文件存放的绝对路径；out_dir为存放输出文件的绝对路径；log.file为程序运行中产生的日志文件名称。程序运行示例：输出文件为两个pool的bam文件：3.SNPcalling脚本2callSNPfromBam.pl调用了samtools软件，将两个混合池中的SNPcall出来。2callSNPfromBam.pl需要5个必须输入