第二代測(cè)序技術(shù)-新一代基因組測(cè)序技術(shù)原理及應(yīng)用課件

NewGenerationsofGenomicSequencingTechnologies&Applications

新一代基因組測(cè)序技術(shù)原理及應(yīng)用

NewGenerationsofGenomicSeqOutline

概要Sequencingtechnology(技術(shù)）Historicaloverview（測(cè)序技術(shù)發(fā)展回顧）Currenttrendsinsequencingtechnology（測(cè)序技術(shù)的走向）2ndGenerationSequencer（第二代測(cè)序儀）NewNext-Generation

-Singlemoleculesequencing(SMDS)（第三代及單分子測(cè)序）Nanoanddirectsequencing(納米及直接測(cè)序技術(shù)）SequencingApplications（應(yīng)用）ConventionalsequencingapplicationsNewapplicationareasofsequencingOutline概要SequencingtechnologDNA測(cè)序主要方法Sequencingbyseparation（分離測(cè)序法）Sanger’schain-terminationmethodMaxam&GilbertchemicalmethodSequencingbysynthesis（合成測(cè)序法)BybaseextensionByligationSequencingbydegradation（降解測(cè)序法)Sequencingbyhybridization（雜交測(cè)序法)Oligo-probesmicroarray（寡核苷酸探針微陣列芯片）+fluorescentlylabeledunknownDNAfragmentsDirectsequencing（直接測(cè)序法）DNA測(cè)序主要方法SequencingbyseparatDNA測(cè)序發(fā)展過程0th-Generation(第零代，1975～1985)Sanger’schain-termination(手工Sanger測(cè)序法）1st-Generation(第一代，1986～2006)Dye-terminatorsequencer（自動(dòng)化熒光標(biāo)記鏈終止測(cè)序法）2nd-Generation(第二代，2006～Present)Sequencingbysynthesisinessemble（DNA鏈合成測(cè)序法）3rd-Generation(第三代，Present~3years)Real-timeandsinglemoleculesequencing（實(shí)時(shí)、單分子合成測(cè)序法）4th-Generation(第四代，in3~5years?)Directsequencing（直接測(cè)序法）DNA測(cè)序發(fā)展過程0th-Generation(第零代，1KeyGenomicsTechnologies

與新一代測(cè)序有關(guān)的主要技術(shù)SouthernDNAhybridizationtechnique（DNA雜交技術(shù)）-1975Sanger’schain-termination（鏈終止法）andMaxam、Gilbert’schemicalDNAsequencingmethods（化學(xué)裂解法）-1977Automatedinsituoligonucleotidesynthesisinstrument（原位寡核苷酸合成儀）-

1980Mullis’sdiscoveryofPCRatCetus(聚合酶鏈?zhǔn)椒磻?yīng)

DNA擴(kuò)增)

–1985KeyGenomicsTechnologies

與新一代KeyGenomicsTechnologiesABI’sautomatedDNAsequencer(全自動(dòng)核酸序列測(cè)定儀問世)

-

1986Affymatrix(Fodor’sgroup)firstgene-chip（原位合成基因芯片的制造）-

19922nd-generationDNAsequenceronmarket（第二代測(cè)序儀問世）-

2006Singlemoleculesequencing（單分子測(cè)序技術(shù)涌現(xiàn)）-

2008～Bioinformatics（生物信息學(xué)）-midof1980s

KeyGenomicsTechnologiesABI’sDriverforFasterandCheaperSequencingTechnology（測(cè)序發(fā)展動(dòng)力）PersonalGenomeProject個(gè)人基因組計(jì)劃

可高速、廉價(jià)、便宜地解讀DNA的下一代測(cè)序技術(shù)將在十年內(nèi)實(shí)現(xiàn)。它變革研究和促使真正個(gè)體化醫(yī)藥時(shí)代到來的潛力告訴我們：我們現(xiàn)在必須要做好準(zhǔn)備了。喬治丘吉爾每一個(gè)人的基因組DriverforFasterandCheaperGenomicSequencingMoore’sLaw

測(cè)序發(fā)展的摩爾定律GenomicSequencingMoore’sLawPicturesTalk

看圖說話

Now1Year2000(CeleraGenomics)

400PicturesTalk

看圖說話Now1Year20Complementarity

各代間的兼容、互補(bǔ)性Complementarity

各代間的兼容、互補(bǔ)性TrendsinNext-GenSequencer

新一代測(cè)序技術(shù)趨勢(shì)Large-scaleandhigh-throughput（大規(guī)模、高通量）Massivelyparallelprocessing（高度平行化）Microarrayofclustersorbeads（固相微陣列）Simultaneoussequencingbysynthesis(平行合成測(cè)序）Opticalmicroscopyfordetection（光學(xué)檢測(cè))Componentminiaturization（裝置微型化）Micro/nano（微納技術(shù)）Microfluidic（微觀流體組件）Simplifiedprotocols（測(cè)序流程的簡(jiǎn)化）

TrendsinNext-GenSequencer

新TechnologyConvergence

多學(xué)科交叉增加并向微納技術(shù)傾斜TechnologyConvergence

多學(xué)科交叉增加ConventionalSequencingTechnologies

傳統(tǒng)測(cè)序技術(shù)

(byLadderFragmentsSeparation)Maxam、Gilbert’schemicalDNAsequencingmethods（化學(xué)裂解測(cè)序法）Sanger’sdideoxy-chainterminationmethod(雙脫氧鏈終止法)Dye-terminationmethod(熒光標(biāo)記鏈終止法）ConventionalSequencingTechno(1)(3)(2)凝膠電泳分離放射自顯影譜放射性同位素標(biāo)記引物4種獨(dú)立聚合反應(yīng)(1)(3)(2)凝膠電泳分離放射自顯影譜放射性同位素標(biāo)記引SequencingwithFluorescentlyTaggedChain-Terminator

熒光標(biāo)記鏈終止測(cè)序法1986LeroyHoodoftheCaliforniaInstituteofTechnologyandcolleaguesannouncethefirstautomatedDNAsequencingmachine1987AppliedBiosystemsInc.putsthefirstautomatedsequencingmachine,basedonHood'stechnology,onthemarketSequencingwithFluorescentlyFourfluorescentlylabeledterminators（4種熒光染料標(biāo)記鏈終止核苷酸）OneDNApolymerizationreaction（1個(gè)聚合反應(yīng)）Replicationproductseparatedbygel-electrophoresis（熒光標(biāo)記鏈終止產(chǎn)物由電泳分離）經(jīng)電泳后各個(gè)熒光譜帶分開，同時(shí)激光檢測(cè)器同步掃描，激發(fā)出的熒光經(jīng)光柵分光后打到CCD攝像機(jī)上同步成像，將信息輸送給電腦進(jìn)行分析和保存熒光測(cè)序儀ABI377有64條泳道FourfluorescentlylabeledterAutomatedDNASequencer

全自動(dòng)測(cè)序儀

毛細(xì)管電泳激發(fā)出的熒光被采集，輸送給電腦進(jìn)行分析和保存熒光標(biāo)記的鏈終止COPY產(chǎn)物AutomatedDNASequencer

全自動(dòng)測(cè)序儀Progressionof1st-GenerationThroughput

第一代儀器測(cè)序通量演變Progressionof1st-Generation1st–

GenerationLimitation

一代測(cè)序儀的局限

LowThroughput（通量低）Time-consumingseparationofchain-terminatedfragments（電泳分離耗時(shí)）Hardtoproducemassivelyparallelsystembasedelectrophoreticseparation（電泳分離不易大規(guī)模平行）HighSequencingCost（成本高）Complexsamplepreparation&handling（樣品準(zhǔn)備處理復(fù)雜）Highreagentconsumption（試劑用量大）Difficulttominiaturize（技術(shù)不易微量化）1st–GenerationLimitation

一代二代合成測(cè)序法的基石

DNA聚合反應(yīng)+ppi+H+二代合成測(cè)序法的基石

DNA聚合反應(yīng)+ppi+H+2nd-GenerationSequencing

Platforms

二代測(cè)序平臺(tái)Illumina/SolexaGenomicAnalyzerSequencebysynthesiswithreversiblefluorescentterminators（可逆性鏈終止合成測(cè)序）Roche/454LifeSciencesGSFLXPyrosequencing（焦磷酸測(cè)序）ABI/AgencourtSOLiDSystemSequencebyligationwithdualbaseencoding（雙堿基編碼連接測(cè)序）2nd-GenerationSequencingPlatWorkflowof2nd-GenSequencing

二代測(cè)序流程FragmentLibraryPreparation(DNA模板文庫制備）RandomPair-endImmobilizationofFragment

（DNA片段固定）Surface,BeadCovalentornon-covalentParallelSequencebySynthesis（平行測(cè)序反應(yīng)）BaseextensionLigationSimultaneous

ImageAcquisition（光學(xué)圖像采集與處理）FluorescenceChemiluminescenceSequenceReadandAssembly（序列拼接、組裝）ClonalAmplification（DNA片段單分子擴(kuò)增）EmulsionPCRPoloniesCycleWorkflowof2nd-GenSequencingSmallfragmentsAsymmetricAdaptorsIllumina/SolexaTechnologySmallfragmentsAsymmetricAdapSinglestrandedDNAfragmentspreparedandattachedtosolidsurface(單鏈片斷固定到載片表面)DNAfragmentsareamplifiedtoformclusters（orpolonies）ofsingle-strandedtemplate(DNA簇群生成)Bridgeamplification

（橋式擴(kuò)增法）Allpoloniesaresimultaneouslysequencedbypolymerizationandfluorescenceimagingcycles（循環(huán)合成反應(yīng)+熒光成像）SolexaSequencingStepsSinglestrandedDNAfragmentsSmallfragments（DNA片段）AsymmetricAdaptors（不對(duì)稱接頭）

FragmentLibraryPrep（模板制備）LigateSmallfragmentsAsymmetricAdapAttachFragmentstoSurface

表面固定FlowCell載體芯片單鏈引物AttachFragmentstoSurface

表面BridgeFormation

“橋”的形成Anchoredprimersandtemplateswithinaradiuswillamplify.PrimaryampliconBridgeFormation

“橋”的形成AnchoreYieldtwocovalentmolecules3’3’BridgeAmplification

“橋”擴(kuò)增Yieldtwocovalentmolecules3’Templatesbecomepermanentlybindtosurface.BridgeDenaturation

“橋”變性TemplatesbecomepermanentlyOne-moleculetemplateformsoneclusterOnlyonesetofprimersareused(B-orR-primer)經(jīng)過30輪擴(kuò)增，每個(gè)單分子得到了1000倍擴(kuò)增，成為單克隆“DNA簇群”

Polonies

聚合酶族群生成One-moleculetemplate經(jīng)過30輪擴(kuò)增，SimultaneousSynthesis

同步鏈合成反應(yīng)引物+DNA聚合酶+4種不同色熒光標(biāo)記的可逆終止核苷酸SimultaneousSynthesis

同步鏈合成反應(yīng)位于堿基3'末端的保護(hù)基團(tuán)被除去，繼續(xù)下一輪反應(yīng)標(biāo)記熒光經(jīng)過掃描進(jìn)行識(shí)別，讀取該次反應(yīng)顏色FluorescentImaging

熒光成像位于堿基3'末端的保護(hù)基團(tuán)被除去，繼續(xù)下一輪反應(yīng)標(biāo)記熒光經(jīng)SecondSynthesis

第二輪合成SecondSynthesis

第二輪合成SecondImaging

第二輪成像SecondImaging

第二輪成像RepeatCycles

循環(huán)重復(fù)RepeatCycles

循環(huán)重復(fù)ProsThroughputisveryhigh–通量高Relativelyinexpensive–相對(duì)便宜ConsShortread–讀長(zhǎng)短ProsandCons（主要優(yōu)缺點(diǎn)）ProsProsandCons（主要優(yōu)缺點(diǎn)）Roche/454LifeSciencesGenomeSequencer試劑液體傳送系統(tǒng)光學(xué)檢測(cè)系統(tǒng)計(jì)算機(jī)系統(tǒng)Roche/454LifeSciencesGenomeSequencingMethodBasedonReal-timePyrophosphate（焦磷酸測(cè)序法）RonaghiM,UhlenM,NyrenPADepartmentofBiochemistryandBiotechnology,TheRoyalInstituteofTechnology,Stockholm,Sweden

Science1998;281:363,365SequencingMethodBasedonSinglestrandedDNAtemplatespreparedandattachedtosurfaceofmagneticbead(DNA單鏈片斷固定在球珠表面)DNAfragmentisamplifiedthroughemPCR

(乳水包PCR擴(kuò)增)

andenriched（篩選）Amplifiedbeadsaredepositedonflowcellwithmicrowells,onebeadperwell（球珠->微反應(yīng)池）Eachbead,fixedinwell,issequencedbycyclingthrough

pyrophosphatechemistryandchemiluminescentimaging（循環(huán)合成反應(yīng)+化學(xué)發(fā)光成像）Roche/454SequencingStepsSinglestrandedDNAtemplatesPyrophosphateChemistry

焦磷酸酶級(jí)聯(lián)化學(xué)反應(yīng)PPi+APS(adenosine5′phosphosulfate)ATPhydrolyzedbyluciferaseusingluciferintoproducelightRemove(d)NTPsandexcessATP由4種酶催化的同一反應(yīng)體系中的化學(xué)發(fā)光反應(yīng)(焦磷酸基團(tuán))(ATP硫酸化酶)

(熒光素酶)

(雙磷酸酶)

BreakATPdown(聚合酶)（dATPS，dTTP，dCTP，dGTP四種核苷三磷酸）NPyrophosphateChemistry

焦磷酸酶級(jí)聯(lián)Roche/454Workflow（工作流程）DNALibraryPrepDNAFragmentendrepairedAsymmetricadaptorsligatedDenatured->sstemplateDNAlibraryEmulsionPCRAmplification

(乳滴PCR擴(kuò)增）TemplateDNAimmobilizedonprimercoatedbeadsthruhybridization(1fragmentoneachbead)ThermocycletoamplifyAmplifiedbeadsenrichedwithstreptavidincoatedmagneticbeadsRoche/454Workflow（工作流程）DNALiEmulsionPCR

（油包水）乳滴PCR擴(kuò)增IVC:Invitrocompartmentalization將PCR反應(yīng)物包被于“油包水”的乳化劑中，PCR擴(kuò)增過程就可以在每一滴乳化劑內(nèi)獨(dú)立進(jìn)行bead.

微型反應(yīng)器

EmulsionPCR

（油包水）乳滴PCR擴(kuò)增IVC:Roche/454WorkflowBeadDepositionOneamplifiedbeadpermicrowellFollowedbyenzymebeadsandpackingbeadsEnzymebeadsSulfurylaseLuciferasePackingbeadshelptokeepDNAbeadinmicrowellPyrosequencing4nucleotidessequentiallyflowinIncorporationofanucleotidereleasesapyrophosphate(PPi)SufurylaseconvertPPiintoATPATPhydrolyzedbyluciferaseusingluciferintoproducelightRoche/454WorkflowBeadDeposiRoche/454WorkflowImageAcquisitionCCDcameracoupledtothepicotiterplateChemiluminescentintensityreflectsnumberofnucleotideincorporatedineachflow;usedtodeterminehomopolymerregionUpto100cyclesrepeatedPost-acqProcessingDenovosequencingResequencingAmpliconvariantanalysisImageProcessingChemiluminescenteventmappedtowellFlowgramgeneratedforeachwellBasecalledRoche/454WorkflowImageAcquiProsThroughputishighrelativeto1st-gen–高通量Longerreadlength–高讀長(zhǎng)ConsReagentcosthigh–消耗試劑貴Difficultywithhomopolymerrun–難以分辨同聚物ProsandCons（主要優(yōu)缺點(diǎn)）ProsProsandCons（主要優(yōu)缺點(diǎn)）ABISOLiD–SequencebyLigationwithDual-baseEncoding

雙基編碼連接酶測(cè)序法ABISOLiD–SequencebyLigatiSinglestrandedDNAtemplatespreparedandattachedtobead(DNA單鏈片斷固定在球珠表面)

DNAfragmentisamplifiedthroughemulsionPCR

(乳滴PCR擴(kuò)增)

andenriched（篩選）Amplifiedbeadsattachedtoglassslidesurface（球珠被固定到玻璃載體表面）Eachbeadissequencedbycyclingthrough

ligationandfluorescenceimaging（循環(huán)連接反應(yīng)+熒光成像）SOLiDSequencingSteps

SinglestrandedDNAtemplatesABISOLiD–LibraryPrepShearedfragmentsaretaggedwithadapters(A1andA2)toeachendABISOLiD–LibraryPrepSheareABISOLiD–EmulsionPCREmulsionPCRperformedusingDNAfragmentsfromlibraryonbeads(μm)coatedwithoneoftheprimers3’-endofamplifiedDNAstrandsmodifiedABISOLiD–EmulsionPCREmulsiABISOLiD–BeadDepositionAmplifiedbeadenrichedonpolystyrenecapturebeadscoatedwithA2adaptor;anybeadcontainingtheextendedproductswillbindpolystyrenebeadthroughitsP2end.ThisincreasethethroughputofbeadswithtargetedDNAfrom30%to80%3’endofenrichedproductmodifiedtoallowcovalentattachmenttoglassslidesurfacerandomlyABISOLiD–BeadDepositionAmpFluorescentOligoOctamerProbes

4組熒光雙堿基編碼寡核苷酸探針Degeneratenucleotides簡(jiǎn)并堿基DinucleotidesFluorescentOligoOctamerProbSequencebyLigationHybridizationandligationofaspecificoligowhose1st&2ndbasesmatchthatofthetemplate連接寡核苷酸探針

SequencebyLigationHybridizatSequencebyLigationDetectionofthespecificfluorescence成像SequencebyLigationDetectionSequencebyLigation保護(hù)未連接鏈SequencebyLigation保護(hù)未連接鏈SequencebyLigationCleavageofallbasestothe5’ofbase5去除熒光標(biāo)記SequencebyLigationCleavageoSequencebyLigation重復(fù)連接反應(yīng)七次SequencebyLigation重復(fù)連接反應(yīng)七次SequencebyLigationPrimerandallligatedportionsaremeltedfromthetemplateanddiscardedNewinitialprimerisusedthatisN-1inlength重啟引物SequencebyLigationPrimerandSequencebyLigationGeneratesanoverlappingdataset重啟后循環(huán)連接反應(yīng)SequencebyLigationGeneratesFullSequenceCoverage

模板片段覆蓋完畢FullSequenceCoverage

模板片段覆蓋完Di-BaseEncodingColorSpace

雙堿基編碼顏色空間解讀Di-BaseEncodingColorSpace

雙SingleColorChange=SequencingErrorTwoColorChange=SNP(單核苷多態(tài)性）ColorSpaceErrorDetectionSingleColorChange=SequenciProsThroughputisveryhigh–高通量Build-inerrordetection–誤差校正ConsShortreadlength–讀長(zhǎng)短ProsandCons（主要優(yōu)缺點(diǎn)）ProsProsandCons（主要優(yōu)缺點(diǎn)）模塊化DNA分析系統(tǒng)的成功研制

（中科院北京基因組研究所/中科院半導(dǎo)體研究所）中國(guó)科學(xué)院重大科研裝備研制項(xiàng)目2011年4月完成項(xiàng)目驗(yàn)收工作焦磷酸測(cè)序法+四工位平臺(tái)達(dá)到國(guó)際主流測(cè)序設(shè)備技術(shù)指標(biāo)：讀長(zhǎng)>500bp模塊化DNA分析系統(tǒng)的成功研制

（中科院北京基因組研究所/中BIGIS-4第二代測(cè)序儀樣機(jī)64BIGIS-系列已達(dá)國(guó)外主流設(shè)備性能指標(biāo)BIGIS-4第二代測(cè)序儀樣機(jī)64BIGIS-系列已達(dá)國(guó)外FluorogenicPyrosequencingin

PDMSMicroreactors

（熒光焦磷酸測(cè)序法）

FluorogenicPyrosequencinginLifeTechnologies’IonTorrentSequencingbysynthesissimilarto454(聚合酶合成測(cè)序法）Non-modifiednucleotidesflowinsequentiallyNon-lightbaseddetection（非光學(xué)檢測(cè)）CMOSchipcontainH+sensitivewellsMonitorthereleaseofhydrogenionduringDNAsynthesiswith,essentially,tinypHmeters–通過跟蹤在聚合成過程中氫離子的釋放LifeTechnologies’IonTorrentSemiconductorSequencingIonProton?SequencerSemiconductorSequencingIonPIonTorrentCMOSChipIonTorrentCMOSChipIonTorrentSequencingIonTorrentSequencingProsCheaptobuild-無需光學(xué)檢測(cè)Reagentcostlow

-無熒光標(biāo)記物ConsLowthroughput

-密度有限D(zhuǎn)ifficultywithhomopolymerrun–釋放離子數(shù)難以分辨ProsandCons（主要優(yōu)缺點(diǎn)）ProsProsandCons（主要優(yōu)缺點(diǎn)）二代測(cè)序流程小結(jié)FragmentLibraryPreparation(DNA模板文庫制備）RandomPair-endImmobilizationofFragment

（DNA片段固定）BeadFlatsurfaceParallelSequenceReaction（平行族群測(cè)序反應(yīng)）PolymeraseLigaseSimultaneous

ImageAcquisition（圖像采集與處理）FluorescenceChemiluminescenceSequenceReadandAssembly（序列拼接、組裝）ClonalAmplification（DNA片段擴(kuò)增）EmulsionPCRBridgeamplificationCycle二代測(cè)序流程小結(jié)FragmentLibraryPrepa二代測(cè)序平臺(tái)比較SebastianJünemann,et.al.2013Volume31Number4NatureBiotechnology二代測(cè)序平臺(tái)比較SebastianJünemann,et2nd-GenerationDrawbacks

二代測(cè)序平臺(tái)的弱點(diǎn)Errorrateincreasewithlengthduetoensembleeffect（群體效應(yīng)所導(dǎo)致的誤差）Incompletechemicalreactions（化學(xué)反應(yīng)效率不完全）Hindranceofpolymeraseincorporation（聚合反應(yīng)缺陷）Deblocking(uncap)ofreversibleterminator（鏈終止解封反應(yīng)）Opticalsignaldetectionerror（光信號(hào)檢測(cè)誤差）Strandsinanensemblenotinsync（失去族群同步性）De-phasinginanensemble->ambiguouslightsignal（族群光信號(hào)失相）Therefore,shortread-length（因而讀序短）2nd-GenerationDrawbacks

二代測(cè)序平NextNext(3rd)-GenSequencing

SingleMoleculeSequencing(SMS)

三代單分子測(cè)序Sequencebysynthesis(SBS)insinglemolecule(單分子合成

)UsingfluorescentlylabelednucleotidesReal-timesinglemoleculefluorescencedetection(單分子熒光實(shí)時(shí)監(jiān)測(cè))Immobilizedpolymerasevs.immobilizedDNAtemplate（聚合酶固定vs.DNA模板固定）NextNext(3rd)-GenSequencingSMS單分子測(cè)序流程

FragmentLibraryPreparation(DNA模板文庫制備）RandomPair-endImmobilizationofFragment

（DNA片段固定）FlatsurfaceParallelSequenceReaction（并行測(cè)序反應(yīng)）PolymeraseSimultaneous

ImageAcquisition（圖像采集與處理）FluorescenceSequenceReadandAssembly（序列拼接、組裝）ClonalAmplification（DNA片段擴(kuò)增）EmulsionPCRBridgeamplificationCycleSMS單分子測(cè)序流程FragmentLibraryPrKeyChallengesofSingleMoleculeMonitoring

SingleMolecule

Detection單分子監(jiān)測(cè)Detectionoffluorescencefromsinglemoleculeinreal-time(對(duì)單分子熒光進(jìn)行實(shí)時(shí)監(jiān)測(cè))

Interferenceofbackgroundfluorescence（背景熒光干擾）Non-specificbindingoffluorescentnucleotideonsurface（熒光標(biāo)記核苷酸與表面的非特異性結(jié)合）Fluorescentlylabelednucleotidesinreactionsolution（漂浮在反應(yīng)液的熒光標(biāo)記物）

KeyChallengesofSingleMolecepifluorescentmicroscope

（落射熒光顯微鏡）mercurylampacooledCCDcameraSingleMoleculeFluorescenceMicroscope（熒光顯微鏡）

camera標(biāo)本接物鏡二色分光鏡

epifluorescentmicroscope（落射熒Useevanescentwavetoilluminateandexcitefluorophoresinarestrictedregionadjacenttotheglass-waterinterface(用隱失波縮小熒光監(jiān)測(cè)范圍于玻水界面)Surfaceboundsinglemoleculescanbemonitoredwithhighsignaltonoise(固相表面結(jié)合的單分子熒光信噪比提高)TotalInternalReflectionFluorescence–TIR

全內(nèi)反射熒光顯微鏡技術(shù)UseevanescentwavetoilluminTIRFSchematics

全內(nèi)反射熒光顯微鏡工作原理顯微鏡載片

水介層

隱失波界面入射光

反射光

TIRFSchematics

全內(nèi)反射熒光顯微鏡工作原理HeliScopeGeneticAnalyzer

tSMS–trueSingleMoleculeSequencingHeliScopeGeneticAnalyzer

tAcyclicprocessinvolvingmultipleroundsofA)incorporationoffluorescentnucleotide（核苷酸合成）

B)washing（核苷酸沖洗）

C)imaging（熒光成像）D)cleavinglabel（去除熒光標(biāo)記）tSMSProcess單分子合成測(cè)序合成沖洗成像去除AcyclicprocessinvolvingmulC-T-A-G-T-G-2.C-G-C-T-A-3.C-A-T-G-CCCTAAGGCTTTAGGC-T-A-G-T-G-2.C-G-C-T-A-3.C-AImagingSystem（熒光成像系統(tǒng)）全內(nèi)反射熒光顯微鏡ImagingSystem（熒光成像系統(tǒng)）全內(nèi)反射熒光顯微VisiGen(ABI)BiotechnologiesSequencebysynthesiswithfluorescenceresonanceenergytransfer(SBS-FRET)(熒光共振能量轉(zhuǎn)移+合成測(cè)序)Polymerase+donorfluorophoreNucleotides+acceptorfluorophoreEnergytransferduringnucleotideincorporationAcceptoremitslightofaparticularwavelengthColoroflightusestoidentifythebaseVisiGen(ABI)BiotechnologiesSForster(Fluorescent)ResonanceEnergyTransfer(FRET)

熒光共振能量轉(zhuǎn)移

Energytransfermechanismbetweentwofluorescentdyes(donorandacceptor)throughlongrangedipoledipoleinteractions(T.F?rster1948)(熒光供體、受體間的能量轉(zhuǎn)移）Onlyhappenwhenthedonorandacceptordyesareincloseproximity,<10nm,creatingaverysmallconfinementoftheacceptorexcitationlight

(近距能量轉(zhuǎn)移）Forster(Fluorescent)Resonanc第二代測(cè)序技術(shù)——新一代基因組測(cè)序技術(shù)原理及應(yīng)用課件OpticalDetectionSystem

光學(xué)探測(cè)系統(tǒng)光譜分離激發(fā)光源全內(nèi)反射熒光OpticalDetectionSystem

光學(xué)探測(cè)系ZERO-MODEWAVEGUIDEs

零模波導(dǎo)

forSingle-MoleculeAnalysisatHighConcentrations

M.J.Levene,J.Korlach,S.W.Turner,M.Foquet,H.G.Craighead,W.W.Webb

CornellUniversity,ClarkHall,Ithaca,NY14853,

USA

Opticalapproachesforobservingthedynamicsofsinglemoleculeshaverequiredpico-tonanomolarconcentrationsoffluorophoreinordertoisolateindividualmolecules.However,manybiologicallyrelevantprocessesoccuratmicromolarligandconcentrations,necessitatingareductionintheconventionalobservationvolumebythreeordersofmagnitude.WeshowthatarraysofZERO-MODEWAVEGUIDEsconsistingofsubwavelengthholesinametalfilmprovideasimpleandhighlyparallelmeansforstudyingsingle-moleculedynamicsatmicromolarconcentrationswithmicrosecondtemporalresolution.WepresentobservationsofDNApolymeraseactivityasanexampleoftheeffectivenessofZERO-MODEWAVEGUIDEsforperformingsingle-moleculeexperimentsathighconcentrations.

Science31January2003:682

ZERO-MODEWAVEGUIDEs

零模波導(dǎo)

forPacificBiosciences

ZeroModeWaveguide

零模波導(dǎo)PacificBiosciences’sSMRTchip

EachchipcontainsthousandsofZMWs.EachZMWisacylindricalholetensofnanometersindiameter,perforatingathinmetal(e.g.aluminum)filmsupportedbyatransparentsubstrate激發(fā)光源PacificBiosciences

ZeroMode

WhentheZMWisilluminatedthroughthetransparentsubstratebylaserlight,thewavelengthofthelightistoolargetopassthroughthewaveguide’saperture.Attenuatedevanescentlightfromtheexcitationbeamdecaysexponentiallyandpenetratesthelower20-30nmofeachwaveguide,creatingadetectionvolumeofonly20zeptoliters(10-21liters).Thisdramaticreductioninthedetectionvolumeprovidestheneeded1000-foldimprovementinrejectionofbackgroundfluorescence.隱失波?<<λ

WhentheZMWisilluminateZMWwithAnchoredPolymeraseSelectiveimmobilizationofpolymerasetothefusedsilicaflooroftheZMWwasachievedbypassivationofthemetalcladdingsurfaceusingpolyphosphonatechemistry,producingenzymedensitycontrastsofglassoveraluminuminexcessof400:1.Yieldsofsingle-moleculeoccupanciesofapproximately30%wereobtainedforarangeofZMWdiameters.PolymeraseDNA固定聚合酶ZMWwithAnchoredPolymeraseSe核苷酸熒光標(biāo)記Usefulinfocanbeobtainedfromsignalpulses核苷酸熒光標(biāo)記Usefulinfocanbeobta物鏡

色彩分離

激發(fā)出的熒光經(jīng)光柵分光后打到單色CCD攝像機(jī)上同步成像熒光監(jiān)測(cè)二色分光鏡

物鏡色彩分離熒光監(jiān)測(cè)二色分光鏡AdvantageofSMS

單分子測(cè)序優(yōu)勢(shì)

Noneedforamplification（不用擴(kuò)增）Highinformationdensity（信息密度高）Theoreticallimitisdiffractionlimitoflight，λ/2（光衍射極限）Errorratestayflatvs.sequencelength（誤差率不隨鏈的延長(zhǎng)增加）Longerreadlength（讀序長(zhǎng)）NodephasingissueduringsynthesisPotentialnewwayofdetectingmodifiednucleotide（提供修飾堿基檢測(cè)新方法）AdvantageofSMS

單分子測(cè)序優(yōu)勢(shì)NoneChallengesforLight-basedSMS

光學(xué)單分子測(cè)序面對(duì)的問題SequencingAccuracy（精確性）Singlemoleculefluorescencedetection（單分子檢測(cè)）Fluorophoreblinking（熒光間斷）Polymerasefidelity（聚合酶保真度）SequencingReadLength（讀長(zhǎng)）Photophysicaldamage（光損害）Photo-bleachingtofluorophore（熒光團(tuán)漂泊）DamagetoDNApolymerase（聚合酶損傷）ChallengesforLight-basedSMSFutureGeneration（4th-Gen）Physicalmethodandmeasurement（物理方法）Visualizingsequencingbymicrospectroscopy（基于電子顯微鏡直接測(cè)序法）Nano-structurebasedmeasurement

(基于納米結(jié)構(gòu)的測(cè)序法）Physicalandchemicalcombinedapproach（物理及化學(xué)相結(jié)合的手段）Stillininfancy（萌芽期）FutureGeneration（4th-Gen）Phys4th-GenerationBenefits

四代優(yōu)勢(shì)Labelfree（無標(biāo)記物）+amplificationfree（無擴(kuò)增）Noneedforopticalimagingsystem（無需光學(xué)顯微鏡）Simpledataprocessingandstorage（數(shù)據(jù)處理簡(jiǎn)易）Lowercost(低成本)+highspeed（高速度）4th-GenerationBenefits

四代優(yōu)勢(shì)LaMicroscopicSequencing（顯微鏡測(cè)序)

SimplyVisualizing

BasesUsingscanningprobemicroscopes（SPM,掃描探針顯微鏡）Atomicforcemicroscope(AFM，原子力顯微鏡

)CoupledwithRamanspectrumScanningtunnelingmicroscope(STM，掃描隧道顯微鏡)Direct-readGeneticSequencingbyZSGeneticswithElectronMicroscopeUsepolymerasewithheavyatomlabeled（重原子標(biāo)記）nucleotidestosynthesisstrandtobedirectlyreadbyEMMicroscopicSequencing（顯微鏡測(cè)序)Solid-StateNanopore（固體納米孔）

IonBeamSculpturedNanoporeinSiliconNitrideElectronBeamPunchedNanopore

onGrapheneSolid-StateNanopore（固體納米孔）

IoBiologicalNanopore（生物納米孔）

Nanoporewithengineeredprotein

(基因工程蛋白質(zhì)改良納米孔)α-hemolysin（溶血素蛋白）MspA（恥垢分枝桿菌孔蛋白）BiologicalNanopore（生物納米孔）

NanKeyChallengesforNanopore-BasedSequencing

納米孔測(cè)序的挑戰(zhàn)DetectionofindividualbasesasDNApassingthrough(分辨不同堿基)ControloftraversingspeedofDNAchainthroughnanopore(DNA鏈穿孔速度控制)KeyChallengesforNanopore-BaNanoporeDetection

CurrentBlockade-阻塞電流NanoporeDetection

CurrentBloNanoporeDetection

ElectronTunneling–遂穿電流NanoporeDetection

ElectronTuDNA鏈納米孔阻塞電流不同的堿基的阻塞電流不同DNA鏈納米孔阻塞電流不同的堿基的阻塞電流不同NanoporeStrandSequencing聚合酶速度制控–生物分子馬達(dá)NanoporeStrandSequencing聚合酶速NanoporeStrandSequencingNanoporeStrandSequencingApplications

應(yīng)用

Applications

應(yīng)用

ComparisonofNGSPlatforms

新一代測(cè)序平臺(tái)比較ComparisonofNGSPlatforms

新一NGSApplicationTargets

新一代代測(cè)序平臺(tái)應(yīng)用對(duì)象Genomicanalysis(DNA)GenomeorDNAsequencingGenomicvariantdiscovery（基因組變異的發(fā)掘）GenomicalterationandmodificationTranscriptomicanalysis(RNA)TranscriptprofilingSmallRNAanalysisBiomarkerdiscoverybyProtona(Protein)Proof-of-conceptworkinsequencingprotein-boundoligonucleotideaptamer(蛋白/核酸適配體)NGSApplicationTargets

新一代代測(cè)序GenomeDenovosequencing（基因組初始測(cè)序）Initialgenerationoflargegenomesequence（基因組原始序列的產(chǎn)生）Productionofreferencegenomesequence(基因組數(shù)據(jù)庫的建立）Wholegenomere-sequence(整基因組再測(cè)序）Discoveryofgenomicvariations（挖掘基因組變異）,e.g.SNP,indels,copynumber,rearrangement,etc.,amongpopulationUnderstandbiologicalconsequencesofvariantsTargetedre-sequence(定向再測(cè)序）SurveygenomicvariantsinregionsofinterestGenomeDenovosequencing（基因組初EukaryoticGenomeSequencingProjects（真核生物基因組）SubmittedtoNCBI;datatabulatedonMarch1,2010EukaryoticGenomeSequencingPDe-novoEukaryoticGenomeSequencingUsingNGSMostlydoneonSolexaand/or454platformDuetoshort-read,capillaryelectrophoresissequencersareoftenusedtofillthegap,e.g.repetitivesequenceDe-novoEukaryoticGenomeSequRe-sequencingofHumanGenomes

人類基因組再測(cè)序Aimtofindgenome-widevariationamongpopulations，e.g.SNP,indel,CN,….Byfar,themostcommonuseofNGSplatformsIlluminaGAhasbecomethedominantplatforminthisareaHighthroughputandrelativelylowruncosts454hastheadvantageoflongerreadbutconsumablecostishigherandhomopolymerissuesBioinformatictoolsforshortreadRe-sequencingofHumanGenomesRe-sequencingofHumanGenomesRe-sequencingofHumanGenomesTranscriptome轉(zhuǎn)錄組

WholeTranscriptomeShotgunSequencingorRNA-seqArevolutionarytoolfortranscriptomicsPriortothis,microarrayisthetoolofchoiceUtilizedeepsequencingcDNAwithNGStostudytranscriptsmRNAtranscriptanalysisDigitalgeneexpressionanalysis(數(shù)碼基因差異表達(dá)）AlternativemRNAspliceforms(非傳統(tǒng)RNA剪接）Discoveryandprofilingofgenome-widenon-codingsmallRNA（非編碼RNA）ShortreadsareidealforstudyofnovelmiRNAandsiRNA(~30baselongwithregulatoryroles)Transcriptome轉(zhuǎn)錄組

WholeTransAdvantageofRNA-seqEliminatetheneedforclonalamplificationEmulsionPCRorbridgeamplificationNopriorknowledgeofgenomesequencerequiredMicroarrayrequiresitforconstructionofprobesDigitalcountingWiderdynamicrangeassequencinggetsdeeperRelativelylowcost(comparingwitholdtechniques)AdvantageofRNA-seqEliminateEpigenomics(表觀基因組）Genome-wideDNA-proteinsinteraction（蛋白/核酸相互作用）Interactionoftranscriptionfactorwithitsdirecttargets（轉(zhuǎn)錄因子）Genomicprofilingofhistonemodification（組蛋白修飾）Genomicprofilingofnucleosomepositions（核小體定位）DNAmethylation（DNA甲基化）Epigenomics(表觀基因組）Genome-wideChIP-seq

ChromatinImmunoprecipatation-DirectSequencing

染色質(zhì)免疫共沉淀-測(cè)序Proteinofinterestcross-linkedwithDNATreatmentwithformaldehydeDNA-proteincomplexsheartogiverisesmallfragmentsUseantibodyspecifictotheproteintoenrichDNA-proteincomplexDNAreleasedfromproteinDNAfragmentssequencedbyNGSChIP-seq

ChromatinImmunoprecAdvantageofChIP-seq

(vs.ChIP-chip)Notlimitedbymicroarraycontent(不受限于微陣列內(nèi)容）Don’tevenneedtohaveadraftgenomeassemblyavailableManyorganismscan,therefore,bestudiedNobiascausedbyprobehybridizationefficiency（無探針雜交偏愛性）Higherresolution（高分辨率）SinglebaseShortreadof35basessufficientAdvantageofChIP-seq

(vs.ChIMethylome(甲基化組)

byNGSCytosinemethylationplaysanessentialroleingenomeregulationProfilingofmethylationsitesinagenomeSodiumbisulfitetreatmentofgenomicDNACytosine->uracil,methylatedcytosinestaysSequencingofSB-treatedgenomicDNAMethylatedC->C,unmethylated->TDeterminemethylationstateofgenomicsitesMethylome(甲基化組)byNGSCytosinMethylomewithReal-timeSingleMolec