NewGenerationsofGenomicSequencingTechnologies&Applications

新一代基因組測序技術原理及應用

NewGenerationsofGenomicSeqOutline-概要Sequencingtechnology(技術）Historicaloverview（測序技術發展回顧）Currenttrendsinsequencingtechnology（測序技術的走向）2ndGenerationSequencer（第二代測序儀）NewNext-Generation

-Singlemoleculesequencing(SMDS)（第三代及單分子測序）Nanoanddirectsequencing(納米及直接測序技術）SequencingApplications（應用）ConventionalsequencingapplicationsNewapplicationareasofsequencingOutline-概要SequencingtechnolMethodsofSequencing

測序主要方法Sequencingbyseparation（分離測序法）Sanger’schain-terminationmethodMaxam&GilbertchemicalmethodSequencingbysynthesis（合成測序法)BybaseextensionByligationSequencingbydegradation（降解測序法)Sequencingbyhybridization（雜交測序法)Oligo-probesmicroarray（寡核苷酸探針微陣列芯片）+fluorescentlylabeledunknownDNAfragmentsDirectsequencing（直接測序法）MethodsofSequencing

測序主要方法SeTechnologicalEvolution

測序發展過程0th-Generation(第零代，1975～1985)Sanger’schain-termination(手工Sanger測序法）1st-Generation(第一代，1986～2006)Dye-terminatorsequencer（自動化熒光標記鏈終止測序法）2nd-Generation(第二代，2006～Present)Sequencingbysynthesisinessemble（DNA鏈合成測序法）3rd-Generation(第三代，Present~3years)Real-timeandsinglemoleculesequencing（實時、單分子合成測序法）4th-Generation(第四代，in3~5years?)Directsequencing（直接測序法）TechnologicalEvolution

測序發展過程RelatedTechnologies

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

1980Mullis’sdiscoveryofPCRatCetus(聚合酶鏈式反應

DNA擴增)

–1985RelatedTechnologies

與新一代測序有關的RelatedTechnologiesABI’sautomatedDNAsequencer(全自動核酸序列測定儀問世)

-

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

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

2006Singlemoleculesequencing（單分子測序技術涌現）-

2008～Bioinformatics（生物信息學）-midof1980s

RelatedTechnologiesABI’sautoDriverforSequencingTechnology

（測序發展動力）PersonalGenomeProject個人基因組計劃

可高速、廉價、便宜地解讀DNA的下一代測序技術將在十年內實現。它變革研究和促使真正個體化醫藥時代到來的潛力告訴我們：我們現在必須要做好準備了。喬治丘吉爾每一個人的基因組DriverforSequencingTechnoloGenomicSequencingMoore’sLaw

測序發展的摩爾定律GenomicSequencingMoore’sLawAPicture’sWorth

看圖說話

Now1Year2000(CeleraGenomics)

400APicture’sWorth

看圖說話Now1YeaComplementarityofTechnologies

各代間的兼容、互補性Technology技術Characters特征Applications功能性PCR產物測序Denovo新測序Re-seq重測序GT1000/100WGSCBC1stGen第一代1.1Gel平板膠﹢﹢﹢﹢﹢NA1.2CE毛細管-四色熒光﹢﹢﹢﹢﹢﹢﹢﹢﹢﹢﹢NA2ndGen新一代2.1微乳液PCRLRNA﹢﹢﹢NA﹢﹢NANASRNA﹢﹢NA﹢﹢﹢NANA2.2高通量/無反應倉NA﹢﹢﹢NA﹢﹢﹢NANA2.3單分子NA﹢﹢﹢NA﹢﹢﹢﹢1000Future下一代3.1化學/納米技術NA﹢﹢﹢NA﹢﹢﹢﹢10003.2納米技術NA﹢﹢﹢NA﹢﹢﹢﹢1003.3納米技術NA﹢﹢﹢NA﹢﹢﹢﹢100ComplementarityofTechnologieTrendsinNext-GenSequencer

新一代測序技術趨勢Large-scaleandhigh-throughput（大規模、高通量）Massivelyparallelprocessing（高度平行化）Microarrayofclustersorbeads（固相微陣列）Simultaneoussequencingbysynthesis(平行合成測序）Opticalmicroscopyfordetection（光學檢測)Componentminiaturization（裝置微型化）Micro/nano（微納技術）Microfluidic（微觀流體組件）Simplifiedprotocols（測序流程的簡化）

TrendsinNext-GenSequencer

新TechnologyConvergence

多學科交叉增加并向微納技術傾斜KeyTechnology關鍵技術

1stGen第一代2ndGen第二代3rdGen第三代4thGen第四代SangerCyclicArraySMSDirectHybridization分子雜交√√√√Enzyme酶介導生化反應√√√

Fluor-Label熒光標記底物√√√

PCR擴增√√

Electrophoresis電泳√

Micro/Nano微納加工

√√√Fluor-Detection熒光信號識別√√√√SMD單分子信號檢測

√√Electro-Signal電信號識別

√√Micro-Fluidics微量液流控制√

√TechnologyConvergence

多學科交叉增加ConventionalSequencingTechnologies

傳統測序技術

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

熒光標記鏈終止測序法1986LeroyHoodoftheCaliforniaInstituteofTechnologyandcolleaguesannouncethefirstautomatedDNAsequencingmachine1987AppliedBiosystemsInc.putsthefirstautomatedsequencingmachine,basedonHood'stechnology,onthemarketSequencingwithFluorescentlyFourfluorescentlylabeledterminators（4種熒光染料標記鏈終止核苷酸）OneDNApolymerizationreaction（1個聚合反應）Replicationproductseparatedbygel-electrophoresis（熒光標記鏈終止產物由電泳分離）經電泳后各個熒光譜帶分開，同時激光檢測器同步掃描，激發出的熒光經光柵分光后打到CCD攝像機上同步成像，將信息輸送給電腦進行分析和保存FluorescenceSequencer熒光測序儀ABI377有64條泳道FourfluorescentlylabeledterAutomatedDNASequencer(CE)

全自動測序儀

毛細管電泳激發出的熒光被采集，輸送給電腦進行分析和保存熒光標記的鏈終止COPY產物AutomatedDNASequencer(CE)

全Progressionof1st-GenerationThroughput

第一代儀器測序通量演變Progressionof1st-Generation1st–

GenerationLimitation

一代測序儀的局限

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

一代ShotgunSequencing

鳥槍測序法ShotgunSequencing

鳥槍測序法DNAPolymerase

合成測序法的基石-DNA聚合反應+ppi+H+DNAPolymerase

合成測序法的基石-DNA聚2nd-GenerationSequencing

Platforms

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

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

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

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

（橋式擴增法）Allpoloniesaresimultaneouslysequencedbypolymerizationandfluorescenceimagingcycles（循環合成反應+熒光成像）IlluminaSequencingStepsSinglestrandedDNAfragmentsSmallfragments（DNA片段）AsymmetricAdaptors（不對稱接頭）

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”擴增Yieldtwocovalentmolecules3’Templatesbecomepermanentlybindtosurface.BridgeDenaturation

“橋”變性TemplatesbecomepermanentlyOne-moleculetemplateformsoneclusterOnlyonesetofprimersareused(B-orR-primer)經過30輪擴增，每個單分子得到了1000倍擴增，成為單克隆“DNA簇群”

Polonies

聚合酶族群生成One-moleculetemplate經過30輪擴增，SimultaneousSynthesis

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

同步鏈合成反應位于堿基3'末端的保護基團被除去，繼續下一輪反應標記熒光經過掃描進行識別，讀取該次反應顏色FluorescentImaging

熒光成像位于堿基3'末端的保護基團被除去，繼續下一輪反應標記熒光經SecondCycleofSynthesis

第二輪合成SecondCycleofSynthesis

第二輪合SecondImaging

第二輪成像SecondImaging

第二輪成像RepeatCycles

循環重復RepeatCycles

循環重復ProsThroughputisveryhigh–通量高Relativelyinexpensive–相對便宜ConsShortread–讀長短ProsandCons

（主要優缺點）ProsProsandCons

（主要優缺點）Roche/454LifeSciencesGenomeSequencer試劑液體傳送系統光學檢測系統計算機系統Roche/454LifeSciencesGenomeSequencingMethodBasedonReal-timePyrophosphate（焦磷酸測序法）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擴增)

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

pyrophosphatechemistryandchemiluminescentimaging（循環合成反應+化學發光成像）Roche/454SequencingStepsSinglestrandedDNAtemplatesPyrophosphateChemistry

焦磷酸酶級聯化學反應PPi+APS(adenosine5′phosphosulfate)ATPhydrolyzedbyluciferaseusingluciferintoproducelightRemove(d)NTPsandexcessATP由4種酶催化的同一反應體系中的化學發光反應(焦磷酸基團)(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

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

(乳滴PCR擴增）TemplateDNAimmobilizedonprimercoatedbeadsthruhybridization(1fragmentoneachbead)ThermocycletoamplifyAmplifiedbeadsenrichedwithstreptavidincoatedmagneticbeadsRoche/454Workflow（工作流程）DNALiEmulsionPCR

（油包水）乳滴PCR擴增IVC:Invitrocompartmentalization將PCR反應物包被于“油包水”的乳化劑中，PCR擴增過程就可以在每一滴乳化劑內獨立進行bead.

微型反應器

EmulsionPCR

（油包水）乳滴PCR擴增IVC:Roche/454WorkflowBeadDepositionOneamplifiedbeadpermicrowellFollowedbyenzymebeadsandpackingbeadsEnzymebeadsSulfurylaseLuciferasePackingbeadshelptokeepDNAbeadinmicrowellPyrosequencing4nucleotidessequentiallyflowinIncorporationofanucleotidereleasesapyrophosphate(PPi)SufurylaseconvertPPiintoATPATPhydrolyzedbyluciferaseusingluciferintoproducelightRoche/454WorkflowBeadDeposiRoche/454WorkflowImageAcquisitionCCDcameracoupledtothepicotiterplateChemiluminescentintensityreflectsnumberofnucleotideincorporatedineachflow;usedtodeterminehomopolymerregionUpto100cyclesrepeatedPost-acqProcessingDenovosequencingResequencingAmpliconvariantanalysisImageProcessingChemiluminescenteventmappedtowellFlowgramgeneratedforeachwellBasecalledRoche/454WorkflowImageAcquiProsThroughputishighrelativeto1st-gen–高通量Longerreadlength–高讀長ConsReagentcosthigh–消耗試劑貴Difficultywithhomopolymerrun–難以分辨同聚物ProsandCons

（主要優缺點）ProsProsandCons

（主要優缺點）ABISOLiD–SequencebyLigationwithDual-baseEncoding

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

DNAfragmentisamplifiedthroughemulsionPCR

(乳滴PCR擴增)

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

ligationandfluorescenceimaging（循環連接反應+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簡并堿基DinucleotidesFluorescentOligoOctamerProbSequencebyLigationHybridizationandligationofaspecificoligowhose1st&2ndbasesmatchthatofthetemplate連接寡核苷酸探針

SequencebyLigationHybridizatSequencebyLigationDetectionofthespecificfluorescence成像SequencebyLigationDetectionSequencebyLigation保護未連接鏈SequencebyLigation保護未連接鏈SequencebyLigationCleavageofallbasestothe5’ofbase5去除熒光標記SequencebyLigationCleavageoSequencebyLigation重復連接反應七次SequencebyLigation重復連接反應七次SequencebyLigationPrimerandallligatedportionsaremeltedfromthetemplateanddiscardedNewinitialprimerisusedthatisN-1inlength重啟引物SequencebyLigationPrimerandSequencebyLigationGeneratesanoverlappingdataset重啟后循環連接反應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(單核苷多態性）ColorSpaceErrorDetectionSingleColorChange=SequenciProsThroughputisveryhigh–高通量Build-inerrordetection–誤差校正ConsShortreadlength–讀長短ProsandCons

（主要優缺點）ProsProsandCons

（主要優缺點）IonTorrentSequencingbysynthesissimilarto454(聚合酶合成測序法）Non-modifiednucleotidesflowinsequentiallyNon-lightbaseddetection（非光學檢測）CMOSchipcontainH+sensitivewellsMonitorthereleaseofhydrogenionduringDNAsynthesiswith,essentially,tinypHmeters–通過跟蹤在聚合成過程中氫離子的釋放IonTorrentSequencingbysynthSemiconductorSequencing

半導體芯片測序IonProton?SequencerSemiconductorSequencing

半導體芯片IonTorrentCMOS芯片IonTorrentCMOS芯片IonTorrentSequencingIonTorrentSequencingProsCheaptobuild-無需光學檢測Reagentcostlow

-無熒光標記物ConsLowthroughput

-密度有限Difficultywithhomopolymerrun–釋放離子數難以分辨ProsandCons（主要優缺點）ProsProsandCons（主要優缺點）NGSMultiplexingwithBarcode

通過多標簽進行多樣本比對NGSMultiplexingwithBarcode

Summaryof2nd-GenSequencing

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

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

ImageAcquisition（圖像采集與處理）FluorescenceChemiluminescenceSequenceReadandAssembly（序列拼接、組裝）ClonalAmplification（DNA片段擴增）EmulsionPCRBridgeamplificationCycleSummaryof2nd-GenSequencing

2nd-GenPlatformComparison

二代測序平臺比較SebastianJünemann,et.al.2013Volume31Number4NatureBiotechnology2nd-GenPlatformComparison

二代2nd-GenerationDrawbacks

二代測序平臺的弱點Errorrateincreasewithlengthduetoensembleeffect（群體效應所導致的誤差）Incompletechemicalreactions（化學反應效率不完全）Hindranceofpolymeraseincorporation（聚合反應缺陷）Deblocking(uncap)ofreversibleterminator（鏈終止解封反應）Opticalsignaldetectionerror（光信號檢測誤差）Strandsinanensemblenotinsync（失去族群同步性）De-phasinginanensemble->ambiguouslightsignal（族群光信號失相）Therefore,shortread-length（因而讀序短）2nd-GenerationDrawbacks

二代測序平NextNext(3rd)-GenSequencing

SingleMoleculeSequencing(SMS)

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

)UsingfluorescentlylabelednucleotidesReal-timesinglemoleculefluorescencedetection(單分子熒光實時監測)Immobilizedpolymerasevs.immobilizedDNAtemplate（聚合酶固定vs.DNA模板固定）NextNext(3rd)-GenSequencingSMS單分子測序流程

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

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

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

SingleMolecule

Detection單分子監測Detectionoffluorescencefromsinglemoleculeinreal-time(對單分子熒光進行實時監測)

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

KeyChallengesofSingleMolecepifluorescentmicroscope

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

camera標本接物鏡二色分光鏡

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

全內反射熒光顯微鏡技術UseevanescentwavetoilluminTIRFSchematics

全內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（去除熒光標記）tSMSProcess單分子合成測序合成沖洗成像去除AcyclicprocessinvolvingmulVisiGen(ABI)BiotechnologiesSequencebysynthesiswithfluorescenceresonanceenergytransfer(SBS-FRET)(熒光共振能量轉移+合成測序)Polymerase+donorfluorophoreNucleotides+acceptorfluorophoreEnergytransferduringnucleotideincorporationAcceptoremitslightofaparticularwavelengthColoroflightusestoidentifythebaseVisiGen(ABI)BiotechnologiesSForster(Fluorescent)ResonanceEnergyTransfer(FRET)

熒光共振能量轉移

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

(近距能量轉移）Forster(Fluorescent)Resonanc新一代基因組測序技術原理及應用課件OpticalDetectionSystem

光學探測系統光譜分離激發光源全內反射熒光OpticalDetectionSystem

光學探測系3rd-Gen:PacificBiosciencesPacBioRSII3rd-Gen:PacificBiosciencesPaWorkflow工作流程Workflow工作流程度長、精確度度長、精確度ZERO-MODEWAVEGUIDEs

零模波導

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

forZeroModeWaveguide

零模波導PacificBiosciences’sSMRTchip

EachchipcontainsthousandsofZMWs.EachZMWisacylindricalholetensofnanometersindiameter,perforatingathinmetal(e.g.aluminum)filmsupportedbyatransparentsubstrate激發光源ZeroModeWaveguide

零模波導Pacifi

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

隱失波WhentheZMWisilluminateZMWwithAnchoredPolymerase

零模波導+固定DNA聚合酶SelectiveimmobilizationofpolymerasetothefusedsilicaflooroftheZMWwasachievedbypassivationofthemetalcladdingsurfaceusingpolyphosphonatechemistry,producingenzymedensitycontrastsofglassoveraluminuminexcessof400:1.Yieldsofsingle-moleculeoccupanciesofapproximately30%wereobtainedforarangeofZMWdiameters.PolymeraseDNA固定聚合酶ZMWwithAnchoredPolymerase

零核苷酸熒光標記Usefulinfocanbeobtainedfromsignalpulses核苷酸熒光標記Usefulinfocanbeobta物鏡

色彩分離

激發出的熒光經光柵分光后打到單色CCD攝像機上同步成像熒光監測二色分光鏡

物鏡色彩分離熒光監測二色分光鏡PacBioRSIIAdvantagesLongreads:averagelengthsof4,200to8,500Highaccuracy:with99%accuracyDetectDNAmodifications:5-methylcytosine,N6-methyladenine,N4-methylcytosine,oxidativedamage.LeastGCbias.Noamplification:noPCRrequired.PacBioRSIIAdvantagesLongreAdvantageofSMS

單分子測序優勢

Noneedforamplification（不用擴增）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增加）Longerreadlength（讀序長）NodephasingissueduringsynthesisPotentialnewwayofdetectingmodifiednucleotide（提供修飾堿基檢測新方法）AdvantageofSMS

單分子測序優勢NoneChallengesforLight-basedSMS

光學單分子測序面對的問題SequencingAccuracy（精確性）Singlemoleculefluorescencedetection（單分子檢測）Fluorophoreblinking（熒光間斷）Polymerasefidelity（聚合酶保真度）SequencingReadLength（讀長）Photophysicaldamage（光損害）Photo-bleachingtofluorophore（熒光團漂泊）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序法）Nano-structurebasedmeasurement

(基于納米結構的測序法）Physicalandchemicalcombinedapproach（物理及化學相結合的手段）Stillininfancy（萌芽期）FutureGeneration（4th-Gen）Phys4th-GenerationBenefits

四代優勢Labelfree（無標記物）+amplificationfree（無擴增）Noneedforopticalimagingsystem（無需光學顯微鏡）Simpledataprocessingandstorage（數據處理簡易）Lowercost(低成本)+highspeed（高速度）4th-GenerationBenefits

四代優勢LaMicroscopicSequencing（顯微鏡測序)

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（重原子標記）

nucleotidestosynthesisstrandtobedirectlyreadbyEMMicroscopicSequencing（顯微鏡測序)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

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

CurrentBlockade-阻塞電流NanoporeDetection

CurrentBloDNA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速度控制Polymerase聚合酶Speedcontrolthrumolecularmotor速度制控–生物分子馬達速度控制Polymerase聚合酶SpeedcontrNanoporeStrandSequencingNanoporeStrandSequencingApplications

應用

Applications

應用

ComparisonofNGSPlatforms

新一代測序平臺比較PlatformInstrumentCostAmplificationGb/RunReadLengthRunTimeCost/MbRoche454$500,00EmulsionPCR0.14009hrs$84SOLiD$595,000EmulsionPCR335-505days$6IlluminaGA$540,000BridgeAmplification1.335-1004days$7Helicos$900,000N/A37328days$0.5PacificBio$695,000N/A3~1100(upto5000)12hrs?ComparisonofNGSPlatforms

新一NGSApplicationTargets

新一代代測序平臺應用對象Genomicanalysis(DNA)GenomeorDNAsequencingGenomicvariantdiscovery（基因組變異的發掘）GenomicalterationandmodificationTranscriptomicanalysis(RNA)TranscriptprofilingSmallRNAanalysisBiomarkerdiscoverybyProtona(Protein)Proof-of-conceptworkinsequencingprotein-boundoligonucleotideaptamer(蛋白/核酸適配體)NGSApplicationTargets

新一代代測序GenomeDenovosequencing（基因組初始測序）Initialgenerationoflargegenomesequence（基因組原始序列的產生）Productionofreferencegenomesequence(基因組數據庫的建立）Wholegenomere-sequence(整基因組再測序）Discoveryofgenomicvariations（挖掘基因組變異）,e.g.SNP,indels,copynumber,rearrangement,etc.,amongpopulationUnderstandbiologicalconsequencesofvariantsTargetedre-sequence(定向再測序）SurveygenomicvariantsinregionsofinterestGenomeDenovosequencing（基因組初EukaryoticGenomeSequencingProjects（真核生物基因組）OrganismGroupCompletedDraftAssemblyInProgressSUMAnimals4137146287Plants32385111Fungi1012093223Protists64964119TOTAL23329388740SubmittedtoNCBI;datatabulatedonMarch1,2010EukaryoticGenomeSequencingPDe-novoEukaryoticGenomeSequencingUsingNGSOrganismGroupStatusGenomeSize(Mb)Platform(s)DepthAiluropodamelanoleucaAnimalsAssembly2460Solexa56xGrosmanniaclavigeraFungiAssembly32.5Solexa,454,andSanger50xVitisviniferaPlantsAssembly500454andSanger11xCucumissativusPlantsAssembly367SolexaandSanger72xGiantPandaAnimalsAssembly2250Solexa73xMostlydoneonSolexaand/or454platformDuetoshort-read,capillaryelectrophoresissequencersareoftenusedtofillthegap,e.g.repetitivesequenceDe-novoEukaryoticGenomeSequRe-sequencingofHumanGenomes

人類基因組再測序Aimtofindgenome-widevariationamongpopulations，e.g.SNP,indel,CN,….Byfar,themostcommonuseofNGSplatformsIlluminaGAhasbecomethedominantplatforminthisareaHighthroughputandrelativelylowruncosts454hastheadvantageoflongerreadbutconsumablecostishigherandhomopolymerissuesBioinformatictoolsforshortreadRe-sequencingofHumanGenomesRe-sequencingofHumanGenomesPlatformIndividualNo.ofRead(Mb)ReadLengthReadCoverageNo.ofRunsEst.Cost(in1000$)SangerJ.C.Venter328007.5x>340,00070,000Roche454J.Watson932507.4x2341,000SOLiDJ.Lupski2383530x375SolexaAnAfricanMale3,6813540x40250AnHanChinese2,9503536x354001stKoreanMale1,64735,7429x152502ndKoreanMale1,91036,88,10628x30200HelicosS.Quake2,7253228x448Re-sequencingofHumanGenomesTranscriptome轉錄組

WholeTranscriptomeShotgunSequencingorRNA-seqArevolutionarytoolfortranscriptomicsPriortothis,microarrayis