中科院bio2000课程-分子生物学-zhu-part1

TranscriptionTranslationGenomicsFunctionalGenomicsProteomicsHigh-ThroughputBiologySystemsBiology Genomics FunctionalGenomics Proteomics GenomicsWhatcanhappenattheDNAlevel? Chromosomestability Chromatin Telomere Deletion/Insertion Rearrangement Mutation MethylationWholeGenomeSequencingBigScience!HistoryinGenomeSequencingStepsindecodingagenome(oldfashion):1)Libraryconstruction2)PhysicalMap(contig)construction3)DNAsequencingstrategies4)SequenceAssembly5)Finishing6)AnnotationMilestones:Saccharomycescerevisiae:1996,12Mbp,cosmidHomosapiens:1990-2003,109bpOryzasativassp.indica/japonicaTheWalkingMethod1. BuildaveryredundantlibraryofBACswithsequencedclone-ends(cheaptobuild)2. Sequencesome“seed”clones3. “Walk”fromseedsusingclone-endstopicklibraryclonesthatextendleft&rightWalking:AnExampleWalkingoffseveralseedsinparallel• Fewsequentialsteps• AdditionalredundantsequencingEfficientInefficientIngeneral,cansequenceagenomein~5walkingsteps,with20%redundantsequencingUsingTwoLibrariesSolution:UseasecondlibraryofsmallclonesMostinefficiencycomesfromclosingasmalloceanwithamuchlargercloneWholeGenomeShotgunSequencingcutmanytimesatrandomgenomeforward-reversepairedreadsplasmids(2–10Kb)cosmids(40Kb)known
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~500bp~500bp1.FindOverlappingReadsCreatelocalmultiplealignmentsfromtheoverlappingreadsTAGATTACACAGATTACTGATAGATTACACAGATTACTGATAGTTACACAGATTATTGATAGATTACACAGATTACTGATAGATTACACAGATTACTGATAGATTACACAGATTACTGATAGTTACACAGATTATTGATAGATTACACAGATTACTGA1.FindOverlappingReads(cont’d)• CorrecterrorsusingmultiplealignmentTAGATTACACAGATTACTGATAGATTACACAGATTACTGATAGTTACACAGATTATTGATAGATTACACAGATTACTGATAGATTACACAGATTACTGAC:20C:35T:30C:35C:40C:20C:35C:0C:35C:40• Scorealignments• AcceptalignmentswithgoodscoresA:15A:25A:40A:25-A:15A:25A:40A:25A:0MakingaSimulatedReadSimulatedreadshaveerrorpatternstakenfromrandomrealreadsERRORIZER
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Feb15,2001Feb16,2001TheHumanGenomeSequencingProjectAMilestoneinContemporaryBiologyAdraftsequenceofthericegenome(OryzasativaL.ssp.indica).Adraftsequenceofthericegenome(OryzasativaL.ssp.japonica).Thegenomesequenceandstructureofricechromosome1Sequenceandanalysisofricechromosome4TheRiceGenomeSequencingProject180926673571045TheNextGenerationSequencing454Solexa/IlluminaSOLiDPacificBioThe454TechnologyinDNASequencingTheInternationalHapMapProjectisamulti-countryefforttoidentifyandcataloggeneticsimilaritiesanddifferencesinhumanbeings.TheHapMapisacatalogofcommongeneticvariantsthatoccurinhumanbeings.Itdescribeswhatthesevariantsare,wheretheyoccurinourDNA,andhowtheyaredistributedamongpeoplewithinpopulationsandamongpopulationsindifferentpartsoftheworldThegoaloftheInternationalHapMapProjectistocomparethegeneticsequencesofdifferentindividualstoidentifychromosomalregionswheregeneticvariantsareshared.IntheinitialphaseoftheProject,geneticdataarebeinggatheredfromfourpopulationswithAfrican,Asian,andEuropeanancestry.Ongoinginteractionswithmembersofthesepopulationsareaddressingpotentialethicalissuesandprovidingvaluableexperienceinconductingresearchwithidentifiedpopulations.*Singlenucleotidepolymorphism**Theregionsoflinkedvariantsareknownashaplotypes***GenerationoftheHapMap FunctionalGenomics Genome-wideK/Oprojects Genome-widegenetaggingprojects EffortsinbuildingORFeomes GeneExpressionprofiling Genome-widemappingofTFbindingsites TheENCODEProjectImportantHistoryoftheYeastGenomeStudies19962001Firsteukaryoticgenomefullysequenced6,200ORFspredicted4042Characterized2244Uncharacterized334Homologs1910UniqueTopicsinThisLecture• Genome-wideK/OProject• Transposonproject• High-throughputproteinlocalization• Protein-proteininteraction(Y2H)• Proteincomplexidentification• Syntheticlethalityscreening• DNA/oligomicroarraysandapplicationGenome-wideGeneDeletion• Homologousyeastrecombinationstrategy• KanMX4selection• BarcodesandtheUpandDownTags• Variousstrainbackground:Diploids:Homovs.HeteroHaploid:MatingTypeaandαStrategyandProtocolStrategyandProtocol6,138Hetero-DiploidStrainsMade• Determinedessentialgene(17%bytetradanalysis)• Deletionphenotypesdeterminefor500genes• Growthfitnessmesuredforsixconditions:Highsalt,sorbitol,galactose,pH8,minimalmediumandnystatintreatment.• ValuablegeneticresoursesTongetal.,Science2001,Vol.294,pp.2364-2368SyntheticLethalityScreeningNetworksofORFsthatAreSyntheticallyLethalOoi,etal.,NatureGenetics35,277-286(2003)The“SLAM”ApproachPathwayPredictionTransposonProjectGenedisruptionMultipleallelesProteinlocalizationGenefunctionvs.growthconditions[Ross-Macdonaldetal.Nature402:413-418(1999)]TRloxLacZURA3tetloxTR3xHATransposonProjectOriginatedfromthe“random”insertioneventbybacterialtransposonsTn3andTn7.Selectablemarker:URA3Reportergene:LacZ(hastobein-frame)Epitope:HAMarkerrecycling:LoxP/CremediatedsystemMutagenesisinvitroSelectionbyTetresistancePlasmidDNApreparationNotIdigestionYeasttransformationandhomologousrecombinationIdentifytheblueguysRecovertheplasmidsandsequenceTransposonMutagenesis[Kumaretal.Genes&Development,16:707-71