molecular biology autumn 19基因组学与比较

现代生物现代生物学的主要研究对象就是基How？当代生物学已进基因(Genome)基因组计基因组计••••人类基因组计划（HumanGenomeProject，HGP）：人类基因组计人类基因组计GenomeHumanGenomeCraigIntl.Francis人类基人类基因组计划的目回答了What的问6国科学6国科学家组成的国家人基因组中心主要研究比•中白宫的庆2000年6白宫的庆2000年6SequencingPubliceffort-Celera-Celera’sviewofSequencingPubliceffort-Celera-Celera’sviewofInternationalUnfaircompetition:CeleradeliveringthesamegoodsbutcanuseICdata,whileICcannotuseCeleradata.Unfaircompetition:ICdeliveringthesamegoodsbutwithstatefunding.遗传图谱(Genetic遗传图谱(Genetic物理图谱(Physical•遗传图又称为连锁图（LinkageMap），是指基因或•••第一代DNA遗传标记是RFLP（RestrictionFragmentLength第二代DNA遗传标记SSLP（SimpleSeqeuceLength微卫星DNA（microsatelliteDNA）。第三代DNA遗传标记SNP（singlenucleotide但更常见的是单个核苷酸的替换，即单核苷酸的多态性。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.鸟枪法序列鸟枪法序列测定技•（shotgunsequencing)技术：随机挑选带有基因序列测定，然后用计算列，导致判断失误。•DNASHEAR&EndReads/±&ENDDNASHEAR&EndReads/±&ENDIndividualsequencingIndividualsequencingreadsarecomparedtoeachotherandwheretheyoverlapcanbeassembledtocreatecontigsKeepaddingindividualsequencingreadstobuildlargerandfewercontigsKeepaddingindividualsequencingreadstobuildlargerandfewercontigsEventuallyallsequencingreadsmergetoasingleconsensussequence(alargecontig)foreachEventuallyallsequencingreadsmergetoasingleconsensussequence(alargecontig)foreachDNADNAsequencingSangerdideoxyNextGenerationsequencing,Roche454IlluminaSolexacyclicalbaseABISOLiDsequencingbyThirdgenerationsequencing,stillindevelopmentSinglemolecule(tetheredDNApolymerase)VisiGen(realtime,FRET-Sanger••DNASanger••DNAisClonedtoaplasmidSeparationReadoutwith•••高通高通量测序技--带来生物革命的新技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 转录本转录本Deepsequencingprovideshigh-throughputevidencefortranscriptsabundanceandtranscriptarchitecture测序取测序取代芯片检测基因表达水ChIP-Seq---TFsandtargetDNACLIP-Seq---RNAbindingproteinsandtargetRNAsequence;SAGE(Serialanalysisofgeneexpression);DGE(DigitalGeneSAGEexperimentsproceedasSAGEexperimentsproceedasIsolatethemRNAofaninputsampleExtractasmallchunkofsequencefromadefinedpositionofeachmRNAmolecule;Linkthesesmallpiecesofsequencetogethertoformalongchain(orconcatemer);Sequencethesechainshigh-throughputDNAProcessthisdatawithacomputertocountthesmallsequencetags.ThreeprinciplesunderlietheSAGEAshortsequencetag(10-14bp)containssufficientinformationtouniquelyidentifyatranscriptprovidedthatthatthetagisobtainedfromauniquepositionwithineachtranscript;Sequencetagscanbelinkedtogethertofromlongserialmoleculesthatcanbeclonedandsequenced;QuantitationofthenumberoftimesaparticulartagisobservedprovidestheexpressionlevelofthecorrespondingsmallsmallRNAidentification(i.e.其它基因组其它基因组计到2006到2006年12月全世界主要基因组计划的进展情不同模式生物基因组的比 尿殖道支原Mycoplasma580肺炎支原不同模式生物基因组的比 尿殖道支原Mycoplasma580肺炎支原Mycoplasma816流感嗜血杆Haemophilus1.8枯草芽孢杆Bacillus4.2大肠杆Escherichia4.6酿酒酵Saccharomyces13 100拟南芥Arabidopsis125果Drosophila165人Homo3约2.5人类基人类基因组草图基本信人人类基因组研究成果表NextStepsonNextStepsonFinishthehumansequenceLarge-scaleidentificationofregulatoryregionsSequencingofadditionallargeCompletingthecatalogueofFromsequenceto人类基人类基因组计HumanGenome基因组学和个性化医ApplicationstoApplicationstoAkeyapplicationofhumangenomeresearchistofinddiseasegenesbypositionalcloningThismethodinvolvesmappingthechromosomalregioncontainingthegenebylinkageanalysisinaffectedfamiliesThehumangenomicsequenceinpublicdatabasesallowsrapididentificationinsilicoofcandidategenes,followedbymutationscreeningofrelevantcandidates,aidedbyinformationongenestructureForamendeliandisorder,agenesearchcannowoftenbecarriedoutinamatterofmonthswithonlyamodestlysized疾病发生疾病发生或疾病易感基健康正常人的DNA序糖尿病患者的DNA序通过科学家们的研究发现，拥有三、四位碱基改变的那些人对于对应疾病表现出比普通人相对而言比乳腺癌和卵巢乳腺癌和卵巢癌是具有家族遗向的，约15-有家族史2009年2009年，Stanford的Quake教授他开发的新测序仪测出了他自己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统比Illumina(Solexa)第二代高速基因分析系统比Illumina(Solexa)SolidIII1品制备（1天），通过合成测序(Sequenceby方法(SequencebyLigation)进行测序，反应中四种碱基同时加入通过检测相邻碱基的2色荧光(2-basecoding)2复杂的乳液PCR，无法自动化，人工操6.实验室要求乳液PCR实验堆环境要求高，需要单独除常规PCR,离心机等设备外，需特殊设备，如beadcounter,hydroShearChiP-Seq,小RNA,甲基化，转录组研究等等无法做新物种测序，绝大多数应用未得，75bp原始数据3x一致准确性ChiP-Seq,小RNA,甲基化，转录组研究等等无法做新物种测序，绝大多数应用未得，75bp原始数据3x一致准确性9.对数据分析读取的是颜色信号而非碱基序列，对服务器的配置要求很高，对数据分析也有10.实验费用2007年初上市，全球占有率~70%.中国约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