鸟枪法测序原理 (2).ppt

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1、RegionalmappingRegionalmappingMinimaltilingpathselectedforsequencing.Regionalmapping9/17/2021>20kbp~300bpMolecularweightmarkerevery5thlaneRestrictionfragmentfingerprinting-BACclonesaregrownin96-wellformat-HindIIIdigest-1%agaroseContigassemblyCloneABCD

2、EFGFPC*OverlapidentificationbyrestrictionpatternsimilaritiesFacilitatedcontigassembly*SangerCentreC.Soderlund,ILongdenandR.Mott******Allrestrictionfragmentswithinacloneselectedforthetilingpathmustbeverifiedbytheirpresenceinoverlappingclones.:vectorfra

3、gments:insertfragmentsBCM-HGSCShotgunSequencingI:RANDOMPHASEBacClone:100-200kbShearedDNA:1.0-2.0kbSequencingTemplates:RandomReadsBCM-HGSCShotgunSequencingII:ASSEMBLYConsensusSequenceGapLowBaseQualitySingleStrandedRegionMis-Assembly(Inverted)BCM-HGSCCo

4、nsensusSequenceGapLowBaseQualitySingleStrandedRegionMis-Assembly(Inverted)BCM-HGSCShotgunSequencingIII:FINISHINGConsensusSequenceGapSingleStrandedRegionMis-Assembly(Inverted)BCM-HGSCShotgunSequencingIII:FINISHINGConsensusSequenceGapMis-Assembly(Invert

5、ed)BCM-HGSCShotgunSequencingIII:FINISHINGConsensusMis-Assembly(Inverted)BCM-HGSCShotgunSequencingIII:FINISHINGBCM-HGSCShotgunSequencingIII:FINISHINGHighAccuracySequence:<1error/10,000basesWholeGenomeShotgunSequencingWholeGenome:3,000MbShearedDNA:1.0-2

6、.0kbSequencingTemplates:RandomReadsBCM-HGSCWholeGenomeShotgunSequencing:AssemblyConsensusSequenceGapLowBaseQualitySingleStrandedRegionMis-Assembly(Inverted)BCM-HGSCWholeGenomeShotgunSequencing:AssemblyConsensusSequenceGapLowBaseQualityBCM-HGSCP/E‘Cele

7、ra’Scheme:10Xcoverageinthreeyears-Contigs1.0-15kb-#Gaps?>100,000?-BaseQualityHighorLow?BCM-HGSC-Mis-Assemblies?-Duplications?-Regionsnotcovered-Regionsverydenselycovered‘Completeanaccurate,highqualitysequenceofthehumangenomebytheendof2003,…….aworkingd

8、raftcanbecompleted…withinthenextthreeyears…’BCM-HGSC‘That(draft)sequencewillbeofloweraccuracyandcontiguity…..…willbeusefulforfindinggenes…andotherfeatures….’BCM-HGSC‘WorkingDraft’SequenceOptions:0.5-1.0X(2reads/kb)-‘Skimming’3.5-4.0X(~9reads/k