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1、外显子组测序目录一、外显子测序简介二、测序深度三、测序平台四、数据分析流程五、数据分析内容六、后期验证外显子测序（也称目标外显子组捕获）是指利用序列捕获技术将全基因组外显子区域DNA捕捉并富集后进行高通量测序的基因组分析方法。是一种选择基因组的编码序列的高效策略，外显子测序相对于基因组重测序成本较低，对研究已知基因的SNP、Indel等具有较大的优势。在人类基因中大约有180,000外显子，占人类基因组的1%，约30MB。NgSB,TurnerEH,RobertsonPD,etal.Targetedcaptureandmassivelyparallelseque
2、ncingof12humanexomes[J].Nature,2009,461(7261):272-276.人类基因组的蛋白编码区域大约包含85%的致病突变。-ChoiM,SchollUI,JiW,etal.GeneticdiagnosisbywholeexomecaptureandmassivelyparallelDNAsequencing[J].ProceedingsoftheNationalAcademyofSciences,2009,106(45):19096-19101.一、外显子测序简介Thesensitivitytodetectheterozygo
3、usvariantswith10readsis78.6%,butincreasesto95.2%at20xandapproximately100%at30xandgreater.[1]Theaveragecoverageofeachbaseinthetargetedregionswas100-fold,and95.3%ofthesebaseswerecoveredsufficientlydeeplyforvariantcalling(≥10×coverage)[2]Exomesequencingproducedahigherlevelofcoveragefort
4、hetargetedsequences(mean,167.50×),slightlyincreasingourabilitytodetectmutationswithVAFsoflessthan10%.[3]ChoiM,SchollUI,JiW,etal.GeneticdiagnosisbywholeexomecaptureandmassivelyparallelDNAsequencing[J].ProceedingsoftheNationalAcademyofSciences,2009,106(45):19096-19101.YanXJ,XuJ,GuZH,et
5、al.ExomesequencingidentifiessomaticmutationsofDNAmethyltransferasegeneDNMT3Ainacutemonocyticleukemia[J].Naturegenetics,2011,43(4):309-315.PlatformsA.GenomicandEpigenomicLandscapesofAdultDeNovoAcuteMyeloidLeukemia[J].NEnglJMed,2013,2013(368):2059-2074.二、测序深度CoveragerateSequencingdepth
6、andcoverageoftheninepairedinitialsequencingsamples.三、测序平台IonProton™IlluminaHiSeq基于IonProton™的外显子测序流程TheboundDNAisisolatedusingstreptavidin-coatedDynabeads®paramagneticbeads,andthenamplifiedandpurified.Thepurified,target-enrichedsampleisthenreturnedtotheIonTorrentsystemworkflowforemul
7、sionPCR,enrichment,andsequencing.ExomesequencingresultsontheIonProton™SystemusingtheIonPI™ChipandtheIonTargetSeq™ExomeKitRawreadsReadsmappedPercentreadsmappedReadsontargetPercentreadsontarget89,782,71987,156,36497.1%68,899,95779.1%MeandepthofcoverageTargetbasesat1xTargetbasesat10xTar
8、getbasesat20
