基因组测序的原理与方法.ppt

大规模基因组测序的原理与方法元素周期表的发现奠定了二十世纪物理、化学研究和发展的基础生命的奥秘蕴藏于“四字天书”之中基因组学的基础理论研究基因组学是一个大学科基因组学是一门大科学基因组与生命之谜测序设备的垄断和高速度换代测序设备发展现状大规模基因组测序的几个支撑技术PCR（聚合酶链式反应）原理Sanger双脱氧末端终止法测序原理大规模基因组测序的两种策略两种大规模基因组测序策略的比较BACbyBACBACbyBAC物理图谱的制作——序列标签位点（STS）作图物理图谱构建的步骤关于文库BAC文库的构建BAC克隆的筛选第二十九页，编辑于星期五：五点十一分。BeijingMap第三十四页，编辑于星期五：五点十一分。共48个ColumnpoolsRowpools“STS-PCR反应池”方案（PoolingProtocol）sheetofsuperpools,platepools,rowpools,columnpools引物OGG1.52对应sp#27,34,45的plate,row,columnpools的筛选结果BACclone确定(+为阳性克隆)引物OGG1.52的Colony-PCR延伸克隆的筛选contig搭建中克隆的错位末端序列步行法（WalkingbyEndSequence）挑取靠近空洞的种子克隆进行末端测序，然后在基因组数据库中进行比对，确定专一性的序列片段作为新的STS路标。最后设计新路标的PCR引物，按照STS—PCR“反应池”方案筛选新的克隆，达到延伸的目的。四、CloneIdentification1、STS-PCR2、BACendsequencing3、Fingerprinting4、FISH霰弹法测序组装与Finishing工作流程图ShotgunSequencingI:RANDOMPHASEShotgunSequencingII:ASSEMBLYConsensusConsensusConsensusConsensusShotgunSequencingIII:FINISHINGBAC-----453F3’sfinishing克隆211B19组装后的序列的错误率为零WholeGenomeShotgunThisbacteriumhasacirculargenomestructurewith2,689,445basepairs,thesecondlargestoneofthermophilesdecodedcompletelytodate.Whatisunderheavenisforall.SunYat-sen,thefatherofmodernChina国际一流测序生产线7万克隆，3000万碱基/天高产出、低成本：$/bp￥/bp美分/bp分/bpContigs:127,550(N50=6,688bp)DeNovoSequencingtheGenomeinBIGSecondgenerationsequencersSecondgenerationsequencersinBIG测序平台SequencingGlossaryGenomeassemblystrategyRecentwholegenomesequencingprojects第七十九页，编辑于星期五：五点十一分。Rawsequencingreadspre-processingIRawsequencingreadspre-processingIIrawdatapre-processImageanalysisandbasecallingQualityControlFastqandQualityDataassessmentI–ReadqualitydistributionLowQualityHighQuality454dinucleotideproportioncheckDataassessmentII–LibraryinsertsizeNumbersofreadswithnon-insertDNA(fulllengthadapter)indifferentinsertsizelibrariesDataassessmentIII–MappingRateSolexaSequencingDataUsagein500bpLibraryDuplicatesdetectionandfilterLanedatausageindifferentsolexalibrary-FiterduplicationreadsAverageReadsperStartPointReadCorrectionCorrectIlluminaGAshortreadsGenomesizeestimationusingKmerHighQualityReadRateafterpreprocessQue