molecular biology autumn 19基因組學與比較

現(xiàn)代生物現(xiàn)代生物學的主要研究對象就是基How？當代生物學已進基因(Genome)基因組計基因組計????人類基因組計劃（HumanGenomeProject，HGP）：人類基因組計人類基因組計GenomeHumanGenomeCraigIntl.Francis人類基人類基因組計劃的目回答了What的問6國科學6國科學家組成的國家人基因組中心主要研究比?中白宮的慶2000年6白宮的慶2000年6SequencingPubliceffort-Celera-Celera’sviewofSequencingPubliceffort-Celera-Celera’sviewofInternationalUnfaircompetition:CeleradeliveringthesamegoodsbutcanuseICdata,whileICcannotuseCeleradata.Unfaircompetition:ICdeliveringthesamegoodsbutwithstatefunding.遺傳圖譜(Genetic遺傳圖譜(Genetic物理圖譜(Physical?遺傳圖又稱為連鎖圖（LinkageMap），是指基因或???第一代DNA遺傳標記是RFLP（RestrictionFragmentLength第二代DNA遺傳標記SSLP（SimpleSeqeuceLength微衛(wèi)星DNA（microsatelliteDNA）。第三代DNA遺傳標記SNP（singlenucleotide但更常見的是單個核苷酸的替換，即單核苷酸的多態(tài)性。到??RFLPWTSSLPRFLPWTSSLPSNPSNPSNPdetectionSNPdetectionorTheThepanelsillustratedetectionoftheA-alleleofanA-to-Gtransition.TheG-allelewouldbedetectedanalogouslyinaparallelreaction.Inpanela,hybridizationwithallele-specificoligonucleotides(ASOs)isshown.TwoshortASOprobesareused,usuallywiththenucleotidecomplementarytotheallelicvariantofthesinglenucleotidepolymorphism(SNP)inthemiddlepositionoftheprobesequence.TheprobesareallowedtobasepairwiththetargetDNAthatcontainstheSNPatconditionsinwhichonlyperfectlymatchedprobe–targethybridsarestable,andhybridsthatcontainamismatchareunstable.Inpanelb,allele-specificprimerextensionisshown.TwoprimersthatannealtotheirtargetsequenceadjacenttotheSNPandhavethenucleotidecomplementarytotheallelicvariantattheir3′-endareusedinprimerextensionreactionscatalysedbyaDNApolymerase.Onlyprimerswithperfectlymatched3′-endswillbeextended.Inpanelc,‘minisequencing’singlenucleotideprimerextensionisshown.OneprimerthatannealstoitstargetsequenceimmediatelyadjacenttotheSNPisextendedbyaDNApolymerasewithasinglenucleotidethatiscomplementarytothenucleotideatthesiteoftheSNP.Theidentityofthenucleotidebywhichtheprimerbecomesextendeddefinesthegenotype.Inpaneld,oligonucleotideligationisshown.Pairsofoligonucleotideprobesthatannealtotheirtargetsequenceadjacenttoeachotherandhaveanallele-specific3′-or5′-nucleotideatthejunctionbetweentheprobesareused.Whentheprobesareperfectlymatchedtotheirtargetsequence,theywillbejoinedbyaligase,whereasamismatchatthejunctioninhibitsligation.Inpanele,invasivecleavageisshown.Pairsofallele-specificoligonucleotideprobesareused,butthesequence5′oftheSNPisunrelatedtothetarget.Inaddition,anupstream(invader)oligonucleotideisusedthatiscomplementarytothesequence5′oftheSNP.Whentheallelespecificoligonucleotideisperfectlymatchedtoitstarget,itisdisplacedattheSNPsitebytheupstreaminvaderoligonucleotide,andtheformedstructureisspecificallyrecognizedandcleavedbyaFLAPendonuclease,whichreleasesthe5′-partoftheInpanelf,restrictionsitecleavageisshown.Restrictionendonucleasesareusedforallele-specificcleavageofthetargetDNAwhenaSNPalterstherecognitionsequencefortheenzyme.Targetmoleculeswithintactrecognitionsiteswillbecleaved,whereastargetmoleculeswithalteredsitesremainuncleaved.Singlenucleotidepolymorphisms(SNPs)arehighlyabundant,Singlenucleotidepolymorphisms(SNPs)arehighlyabundant,andareestimatedtooccurat1outofevery1,000basesinthehumangenome.SNPsinthecodingregionsofgenesthatalterthefunctionorstructureoftheencodedproteinsareanecessaryandsufficientcauseofmostoftheknownrecessivelyordominantlyinheritedmonogenicdisorders.TheseSNPsareroutinelyanalysedfordiagnosticHowever,mostSNPsarelocatedinnon-codingregionsofthegenome,andhavenodirectknownimpactonthephenotypeofanindividual.TheseSNPsareusefulasmarkersinpopulationgeneticsandevolutionarystudies.鳥槍法序列鳥槍法序列測定技?（shotgunsequencing)技術：隨機挑選帶有基因序列測定，然后用計算列，導致判斷失誤。?DNASHEAR&EndReads/±&ENDDNASHEAR&EndReads/±&ENDIndividualsequencingIndividualsequencingreadsarecomparedtoeachotherandwheretheyoverlapcanbeassembledtocreatecontigsKeepaddingindividualsequencingreadstobuildlargerandfewercontigsKeepaddingindividualsequencingreadstobuildlargerandfewercontigsEventuallyallsequencingreadsmergetoasingleconsensussequence(alargecontig)foreachEventuallyallsequencingreadsmergetoasingleconsensussequence(alargecontig)foreachDNADNAsequencingSangerdideoxyNextGenerationsequencing,Roche454IlluminaSolexacyclicalbaseABISOLiDsequencingbyThirdgenerationsequencing,stillindevelopmentSinglemolecule(tetheredDNApolymerase)VisiGen(realtime,FRET-Sanger??DNASanger??DNAisClonedtoaplasmidSeparationReadoutwith???高通高通量測序技--帶來生物革命的新技transformstoday’sbiologyThenextgenerationtechnologiesgeneratehundredsofmillionstobillionsofsmallsequencereadsatonetime.454pyrosequencing(introducedin2005,generatingmillionsof200-400bpreadsin2009),Solexasystem(introducedin2006,generatinghundredsofmillionsof50-100bpreadsin2009)SOLiDsystem(introducedin2007,generatingbillionsof50bpreadsin2009).Thesemethodshavereducedthecostfrom$0.01/basein2004tonearly$0.0001/basein2006,increasedthesequencingcapacityfrom1Mb/machine/dayin2004tomorethan5Gbbases/machine/dayin2009.Roche/Roche/454:GenomeSequencerRoche/GenomeSequencerFLX100Mb/runGenomesequencinginmicrofabricatedhigh-densitypicolitrereactorsMargulies,M.Genomesequencinginmicrofabricatedhigh-densitypicolitrereactorsMargulies,M.Eghold,M.etNature.2005AAsectionofPyrosequencingRoche/Roche/454:GenomeSequencer???????RealTimeSequencingbySynthesisChemiluminescencedetectioninpicotiterplatesAmplification:emulsionPCRupto400,000reads/onaverage250bases/readupto100Mb/runIllumina/Illumina/Solexa:GeneticIllumina/Solexa2000Mb/runReversibleReversiblebasedsequencingSecondFluorescentSecondFluorescentBaseBasecallingfromrawdata:theidentityofeachbaseofaclusterisreadofffromsequentialimages.Illumina/Solexa:Illumina/Solexa:Genetic??????????RealTimeSequencingbySynthesisClonalSingleMoleculeArrayAmplification:bridgingPCR60-330millionreads/runupto35-150bases/read2-33Gb/run8channels,5-40millreads/channelFluorescentlabelsReversible3‘OH5daysforIllumina/Solexa2000Mb/runABISOLiD/LifeAppliedstems，3000MbABISOLiD/LifeAppliedstems，3000Mb/ABISOLiD–ABISOLiD–SequencebyOligonucleotideLigationwithDual-baseEncoding??Substrateattachment;dibaseMakesequencinglibrarybyshearingandadapterAttachDNAfragmentstobeadsandamplifypoloniesinemulsionAttachbeadsto??Theseprobesconsistofeightbaseswithaligationsiteatthe3’end,afluorescentdyeatthe5’endandacleavagesitebetweenthefifthandsixthnucleotide;theTheseprobesconsistofeightbaseswithaligationsiteatthe3’end,afluorescentdyeatthe5’endandacleavagesitebetweenthefifthandsixthnucleotide;thefirstthreenucleotidesaredegeneratebases(N)whichcanbeanyofthefournucleotidebases(i.e.,A,C,T,orG).Thelastthree(Z)areuniversalbaseswhichcanpairwithanyofthefournucleotidebases.Theremainingcentraltwobasesarethebasisof2-baseencoding.Ingeneral,calculatingallthepossibilities,therewillbe1024octamerprobes,4dyeseachrepresenting4dinucleotides,and256probesperdye.SOLiD:SequencingSOLiD:SequencingligationFullFullSequenceSOLiD:SOLiD:DataCollectionandImage-Step6,DecodingData:Fordecodingthedata,whicharerepresentedascolors,wemustfirstknowtwoimportantfactors.First,wemustknowthateachcolorindicatestwobases.Second,weneedtoknowoneofthebasesinthesequence:thisbaseisincorporatedinthesequenceinthelast(fifth)roundofstep5.Thisknownbaseisthelastnucleotideofthe3’-endoftheknownP1.Therefore,sinceeachcolorrepresentstwonucleotidesinwhichthesecondbaseofeachdinucleotideunitconstitutesthefirstbaseofthefollowingdinucleotide,knowingjustonebaseinthesequencewillleadustointerpretthewholesequence(Figure2).[13]ThesequencingThesequencingstepisbasicallycomposedoffiveroundsandeachroundconsistsofabout5-7cycles.EachroundbeginswiththeadditionofaP1-complementaryuniversalprimer.Thisprimerhas,forexample,nnucleotidesandits5’-endmatchesexactlywiththe3’-endoftheP1.Ineachcycle,8-merprobes(1024probes)areaddedandligatedaccordingtotheirfourthandfifthbases.Then,theremainingunboundprobesarewashedout,thefluorescentsignalfromtheboundprobeismeasured,andtheboundprobeiscleavedbetweenitsfifthandsixthnucleotide.Finallytheprimerandprobesareallresetforthenextround.Inthenextroundanewuniversalprimerannealsthepositionn-1(its5’-endmatchestothebaseexactlybeforethe3’-endoftheP1)andthesubsequentcyclesarerepeatedsimilartothefirstround.Theremainingthreeroundswillbeperformedwithnewuniversalprimersannealingpositionsn-2,n-3andn-4relativetothe3'-endofP1.ABISOLiD/Life?????????RealTimeSequencingbyLigationEmulsionABISOLiD/Life?????????RealTimeSequencingbyLigationEmulsionPCRandBeadsonslides85-600millionreads/run3-30Gb/rundualfluorescent8individualchannels/flowcell2flowcells/run14daysfor3000Mb/Cyclic-array??DNACyclic-array??DNAisAdaptorsligatedtoSeveralpossibleprotocolsyieldarrayofPCREnyzmaticextensionwithfluorescentlytaggedCyclicreadoutbyimagingthearray.???Emulsion?Fragments,Emulsion?Fragments,withadaptors,arePCRamplifiedwithinawaterdropinoil.OneprimerisattachedtothesurfaceofaUsedby454,Polonatorand????DNA??DNAfragmentsareflankedwithAflatsurfacecoatedwithtwotypesofprimers,correspondingtotheadaptors.Amplificationproceedsincycles,withoneendofeachbridgetetheredtothesurface.Usedby??“Next“NextGeneration”SequencingTechnologies:RateLimitingFactorsFrontend:MakingthesequencingBackend:Bioinformaticstomakesenseofthe“sequencetsunami”---essemblySingleMoleculeSequencingTechnologies:3rdArrayoftetheredDNApolymeraseBoundtotemplatestrand+Cyclicalbaseaddition(similartoRealtime,imagingFRETHopefulprediction:1Mb/HeliScopeGenetictSMSHeliScopeGenetictSMS–trueSingleMolecule?moleculedetectionExpensive?PacificAGenerationPacificAGenerationSequencingReal-TimeDNASequencingfromSinglePolymerase????150bpcircular~93%raw15xcoverage99.3%accuracyStillearlydaysEidetalFutureFutureGeneration（4th-eg.Nanopore-basedComparisonofComparisonofexistingExamplesofExamplesofApplicationsof“NextGeneration”SequencingTechnologiesBestfor“re-sequencing”,i.e.,aligninggeneratedsequencetoareferenceNextgenerationDNAtechnologiesmayreplacemicroarraysforsomeShendure&JiHumanHumanHapMap?Ingeneticepidemiology,?Ingeneticepidemiology,agenome-wideassociationstudy(GWAorGWAS),alsoknownaswholegenomeassociationstudy(WGAstudy,WGAS),isanexaminationofallormostofthegenesofdifferentindividualsofaparticularspeciestoseehowmuchthegenesvaryfromindividualto?Agenome-wideassociationstudyisanapproachthatinvolvesscanningmarkersacrossgenome(≈0.5Mor1M)ofmanypeople(≈2K)tofindgeneticvariationsassociatedwithaparticulardisease.AlargenumberofsubjectsareneededassociationsbetweenSNPsandcausalvariantsareexpectedtolowoddsratios,typicallybelowInordertoobtainareliablesignal,giventheverylargenumberofteststhatarerequired,associationsmustshowahighlevelofsignificancetosurvivethemultipletestingcorrectionSuchstudiesareparticularlyusefulinfindinggeneticvariationsthatcontributetocommon,complexdiseases??WhatisWhatisaDay2SectionWhyareWhyaresuchstudiespossibleThecompletionoftheHumanGenomeProjectin2003andtheInternationalHapMapProjectin2005,researchersnowhaveasetofresearchtoolsthatmakeitpossibletofindthegeneticcontributionstocommondiseasesDay2SectionWhathaveGWASWhathaveGWAS?In2005,itwaslearnedthroughGWASthatage-maculardegenerationisassociatedwithvariationinthegeneforcomplementfactorH,whichproducesaproteinthatregulatesinflammation(Kleinetal.(2005)Science,308,385–In2007,theWellcomeTrustCase-ControlConsortium(WTCCC)carriedoutGWASforthediseasescoronaryheartdisease,type1diabetes,type2diabetes,rheumatoidarthritis,Crohn'sdisease,bipolardisorderandhypertension.Thisstudywassuccessfulinuncoveringmanynewdiseasegenesunderlyingthesediseases.?Day2SectionExamplesofAssociationscanofExamplesofAssociationscanof14,500nonsynonymousSNPsinfourdiseasesidentifiesautoimmunityvariants.NatGenet.2007Genome-wideassociationstudyof14,000casesofsevencommondiseasesand3,000sharedcontrols.WellcomeTrustCaseControlConsortiumNature.2007;447;661-78GenomewideassociationanalysisofcoronaryarterySamanietal.NEnglJMed.2007;357;443-SequencevariantsintheautophagygeneIRGMandmultipleotherreplicatinglocicontributetoCrohn'sdiseasesusceptibility.Parkesetal.NatGenet.2007;39;830-2Robustassociationsoffournewchromosomeregionsfromgenome-wideanalysesoftype1diabetes.Toddetal.NatGenet.2007;39;857-64AcommonvariantintheFTOgeneisassociatedwithbodymassindexandpredisposestochildhoodandadultobesity.Fraylingetal.Science.2007;316;889-94Replicationofgenome-wideassociationsignalsinUKsamplesrevealsrisklocifortype2diabetes.Zegginietal.Science.2007;316;1336-41Scottetal.(2007)Agenome-wideassociationstudyoftype2diabetesinFinnsdetectsmultiplesusceptibilityvariants.Science,316,1341–1345.????????Day2SectionWhenweWhenweshoulduseInbredorganismsvsEnoughindependentlinesformaskingbackgroundDifferenceoftissueMetagenomescomplexSCIENCE315:1781MetagenomescomplexSCIENCE315:1781(30MARCH 轉(zhuǎn)錄本轉(zhuǎn)錄本Deepsequencingprovideshigh-throughputevidencefortranscriptsabundanceandtranscriptarchitecture測序取測序取代芯片檢測基因表達水ChIP-Seq---TFsandtargetDNACLIP-Seq---RNAbindingproteinsandtargetRNAsequence;SAGE(Serialanalysisofgeneexpression);DGE(DigitalGeneSAGEexperimentsproceedasSAGEexperimentsproceedasIsolatethemRNAofaninputsampleExtractasmallchunkofsequencefromadefinedpositionofeachmRNAmolecule;Linkthesesmallpiecesofsequencetogethertoformalongchain(orconcatemer);Sequencethesechainshigh-throughputDNAProcessthisdatawithacomputertocountthesmallsequencetags.ThreeprinciplesunderlietheSAGEAshortsequencetag(10-14bp)containssufficientinformationtouniquelyidentifyatranscriptprovidedthatthatthetagisobtainedfromauniquepositionwithineachtranscript;Sequencetagscanbelinkedtogethertofromlongserialmoleculesthatcanbeclonedandsequenced;QuantitationofthenumberoftimesaparticulartagisobservedprovidestheexpressionlevelofthecorrespondingsmallsmallRNAidentification(i.e.其它基因組其它基因組計到2006到2006年12月全世界主要基因組計劃的進展情不同模式生物基因組的比 尿殖道支原Mycoplasma580肺炎支原不同模式生物基因組的比 尿殖道支原Mycoplasma580肺炎支原Mycoplasma816流感嗜血桿Haemophilus1.8枯草芽孢桿Bacillus4.2大腸桿Escherichia4.6釀酒酵Saccharomyces13 100擬南芥Arabidopsis125果Drosophila165人Homo3約2.5人類基人類基因組草圖基本信人人類基因組研究成果表NextStepsonNextStepsonFinishthehumansequenceLarge-scaleidentificationofregulatoryregionsSequencingofadditionallargeCompletingthecatalogueofFromsequenceto人類基人類基因組計HumanGenome基因組學和個性化醫(yī)ApplicationstoApplicationstoAkeyapplicationofhumangenomeresearchistofinddiseasegenesbypositionalcloningThismethodinvolvesmappingthechromosomalregioncontainingthegenebylinkageanalysisinaffectedfamiliesThehumangenomicsequenceinpublicdatabasesallowsrapididentificationinsilicoofcandidategenes,followedbymutationscreeningofrelevantcandidates,aidedbyinformationongenestructureForamendeliandisorder,agenesearchcannowoftenbecarriedoutinamatterofmonthswithonlyamodestlysized疾病發(fā)生疾病發(fā)生或疾病易感基健康正常人的DNA序糖尿病患者的DNA序通過科學家們的研究發(fā)現(xiàn)，擁有三、四位堿基改變的那些人對于對應疾病表現(xiàn)出比普通人相對而言比乳腺癌和卵巢乳腺癌和卵巢癌是具有家族遺向的，約15-有家族史2009年2009年，Stanford的Quake教授他開發(fā)的新測序儀測出了他自己2009年2009年The“$10,000The“$10,000humangenomesequencing”Tothefirstteamthatcanbuildadeviceanduseittosequence:100humangenomeswithin10daysorAccuracy:atmost1errorper100,000Accuratecoverageofatleast98%oftheRecurringcostofnomorethan$10,000(US)perPrize:$10Deadline:12:01AMPST,October4,Donors:XFoundation,J.CraigVenterPersonalGenomeMachinePersonalGenomeMachinethePersonalGenomeMachine(PGM),thesilicon-baseddeviceisthesmallestandcheapestDNAdecoderevertohitthemarket.Itcanread10millionlettersofgeneticcode,withahighdegreeofaccuracy,injusttwohours.UnlikeexistingDNAscannersthesizeofmainframesandservers,itfitsonatabletopandsellsforonly$50,000,one-tenththepriceofmachinesalreadyoutForthefirsttimeeveryscientist,localhospitalandcollegewillbeabletoaffordone.IfthePGMtakesoffandregulatorslethim,yourfamilydoctorcouldbuyone--andsocouldyou,if,say,youwantedtoseehowfastthatthinggrowinginyourfridgeismutating.FrancisFrancis“Alwaysaskthebigger,theCrickatSalkHowHowdidflowersevolve?DarwincalledHowHowiscellororganpolarityHowwasdevelopmentevolved?---evo-HowHowtoknowwhentostopHowisepigeneticscausedand第二代高速基因分析系統(tǒng)比Illumina(Solexa)第二代高速基因分析系統(tǒng)比Illumina(Solexa)SolidIII1品制備（1天），通過合成測序(Sequenceby方法(SequencebyLigation)進行測序，反應中四種堿基同時加入通過檢測相鄰堿基的2色熒光(2-basecoding)2復雜的乳液PCR，無法自動化，人工操6.實驗室要求乳液PCR實驗堆環(huán)境要求高，需要單獨除常規(guī)PCR,離心機等設備外，需特殊設備，如beadcounter,hydroShearChiP-Seq,小RNA,甲基化，轉(zhuǎn)錄組研究等等無法做新物種測序，絕大多數(shù)應用未得，75bp原始數(shù)據(jù)3x一致準確性ChiP-Seq,小RNA,甲基化，轉(zhuǎn)錄組研究等等無法做新物種測序，絕大多數(shù)應用未得，75bp原始數(shù)據(jù)3x一致準確性9.對數(shù)據(jù)分析讀取的是顏色信號而非堿基序列，對服務器的配置要求很高，對數(shù)據(jù)分析也有10.實驗費用2007年初上市，全球占有率~70%.中國約50Real-TimeDNAReal-TimeDNASequencingfromSinglePolymerasePrincipleofsingle-molecule,real-timeDNA(A)Experimentalgeometry.AsinglemoleculeofDNAtemplate-bound29DNApolymeraseisimmobilizedatthebottomofaZMW,whichisilluminatedfrombelowbylaserlight.TheZMWnanostructureprovidesexcitationconfinementinthezeptoliter(10–21liter)regime,enablingdetectionofindividualphospholinkednucleotidesubstratesagainstthebulksolutionbackgroundastheyareincorporatedintotheDNAstrandbythepolymerase.(B)SchematiceventsequenceofthephospholinkeddNTPincorporationcycle,withacorrespondingexpectedtimetraceofdetectedfluorescenceintensityfromtheZMW.(1)Aphospholinkednucleotideformsacognateassociationwiththetemplateinthepolymeraseactivesite,(2)causinganelevationofthefluorescenceoutputonthecorrespondingcolorchannel.(3)Phosphodiesterbondformationliberatesthedye-linker-pyrophosphateproduct,whichdiffusesoutoftheZMW,thusending