第二代测序技术-新一代基因组测序技术原理及应用课件

NewGenerationsofGenomicSequencingTechnologies&Applications

新一代基因组测序技术原理及应用

NewGenerationsofGenomicSeqOutline

概要Sequencingtechnology(技术）Historicaloverview（测序技术发展回顾）Currenttrendsinsequencingtechnology（测序技术的走向）2ndGenerationSequencer（第二代测序仪）NewNext-Generation

-Singlemoleculesequencing(SMDS)（第三代及单分子测序）Nanoanddirectsequencing(纳米及直接测序技术）SequencingApplications（应用）ConventionalsequencingapplicationsNewapplicationareasofsequencingOutline概要SequencingtechnologDNA测序主要方法Sequencingbyseparation（分离测序法）Sanger’schain-terminationmethodMaxam&GilbertchemicalmethodSequencingbysynthesis（合成测序法)BybaseextensionByligationSequencingbydegradation（降解测序法)Sequencingbyhybridization（杂交测序法)Oligo-probesmicroarray（寡核苷酸探针微阵列芯片）+fluorescentlylabeledunknownDNAfragmentsDirectsequencing（直接测序法）DNA测序主要方法SequencingbyseparatDNA测序发展过程0th-Generation(第零代，1975～1985)Sanger’schain-termination(手工Sanger测序法）1st-Generation(第一代，1986～2006)Dye-terminatorsequencer（自动化荧光标记链终止测序法）2nd-Generation(第二代，2006～Present)Sequencingbysynthesisinessemble（DNA链合成测序法）3rd-Generation(第三代，Present~3years)Real-timeandsinglemoleculesequencing（实时、单分子合成测序法）4th-Generation(第四代，in3~5years?)Directsequencing（直接测序法）DNA测序发展过程0th-Generation(第零代，1KeyGenomicsTechnologies

与新一代测序有关的主要技术SouthernDNAhybridizationtechnique（DNA杂交技术）-1975Sanger’schain-termination（链终止法）andMaxam、Gilbert’schemicalDNAsequencingmethods（化学裂解法）-1977Automatedinsituoligonucleotidesynthesisinstrument（原位寡核苷酸合成仪）-

1980Mullis’sdiscoveryofPCRatCetus(聚合酶链式反应

DNA扩增)

–1985KeyGenomicsTechnologies

与新一代KeyGenomicsTechnologiesABI’sautomatedDNAsequencer(全自动核酸序列测定仪问世)

-

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

19922nd-generationDNAsequenceronmarket（第二代测序仪问世）-

2006Singlemoleculesequencing（单分子测序技术涌现）-

2008～Bioinformatics（生物信息学）-midof1980s

KeyGenomicsTechnologiesABI’sDriverforFasterandCheaperSequencingTechnology（测序发展动力）PersonalGenomeProject个人基因组计划

可高速、廉价、便宜地解读DNA的下一代测序技术将在十年内实现。它变革研究和促使真正个体化医药时代到来的潜力告诉我们：我们现在必须要做好准备了。乔治丘吉尔每一个人的基因组DriverforFasterandCheaperGenomicSequencingMoore’sLaw

测序发展的摩尔定律GenomicSequencingMoore’sLawPicturesTalk

看图说话

Now1Year2000(CeleraGenomics)

400PicturesTalk

看图说话Now1Year20Complementarity

各代间的兼容、互补性Complementarity

各代间的兼容、互补性TrendsinNext-GenSequencer

新一代测序技术趋势Large-scaleandhigh-throughput（大规模、高通量）Massivelyparallelprocessing（高度平行化）Microarrayofclustersorbeads（固相微阵列）Simultaneoussequencingbysynthesis(平行合成测序）Opticalmicroscopyfordetection（光学检测)Componentminiaturization（装置微型化）Micro/nano（微纳技术）Microfluidic（微观流体组件）Simplifiedprotocols（测序流程的简化）

TrendsinNext-GenSequencer

新TechnologyConvergence

多学科交叉增加并向微纳技术倾斜TechnologyConvergence

多学科交叉增加ConventionalSequencingTechnologies

传统测序技术

(byLadderFragmentsSeparation)Maxam、Gilbert’schemicalDNAsequencingmethods（化学裂解测序法）Sanger’sdideoxy-chainterminationmethod(双脱氧链终止法)Dye-terminationmethod(荧光标记链终止法）ConventionalSequencingTechno(1)(3)(2)凝胶电泳分离放射自显影谱放射性同位素标记引物4种独立聚合反应(1)(3)(2)凝胶电泳分离放射自显影谱放射性同位素标记引SequencingwithFluorescentlyTaggedChain-Terminator

荧光标记链终止测序法1986LeroyHoodoftheCaliforniaInstituteofTechnologyandcolleaguesannouncethefirstautomatedDNAsequencingmachine1987AppliedBiosystemsInc.putsthefirstautomatedsequencingmachine,basedonHood'stechnology,onthemarketSequencingwithFluorescentlyFourfluorescentlylabeledterminators（4种荧光染料标记链终止核苷酸）OneDNApolymerizationreaction（1个聚合反应）Replicationproductseparatedbygel-electrophoresis（荧光标记链终止产物由电泳分离）经电泳后各个荧光谱带分开，同时激光检测器同步扫描，激发出的荧光经光栅分光后打到CCD摄像机上同步成像，将信息输送给电脑进行分析和保存荧光测序仪ABI377有64条泳道FourfluorescentlylabeledterAutomatedDNASequencer

全自动测序仪

毛细管电泳激发出的荧光被采集，输送给电脑进行分析和保存荧光标记的链终止COPY产物AutomatedDNASequencer

全自动测序仪Progressionof1st-GenerationThroughput

第一代仪器测序通量演变Progressionof1st-Generation1st–

GenerationLimitation

一代测序仪的局限

LowThroughput（通量低）Time-consumingseparationofchain-terminatedfragments（电泳分离耗时）Hardtoproducemassivelyparallelsystembasedelectrophoreticseparation（电泳分离不易大规模平行）HighSequencingCost（成本高）Complexsamplepreparation&handling（样品准备处理复杂）Highreagentconsumption（试剂用量大）Difficulttominiaturize（技术不易微量化）1st–GenerationLimitation

一代二代合成测序法的基石

DNA聚合反应+ppi+H+二代合成测序法的基石

DNA聚合反应+ppi+H+2nd-GenerationSequencing

Platforms

二代测序平台Illumina/SolexaGenomicAnalyzerSequencebysynthesiswithreversiblefluorescentterminators（可逆性链终止合成测序）Roche/454LifeSciencesGSFLXPyrosequencing（焦磷酸测序）ABI/AgencourtSOLiDSystemSequencebyligationwithdualbaseencoding（双碱基编码连接测序）2nd-GenerationSequencingPlatWorkflowof2nd-GenSequencing

二代测序流程FragmentLibraryPreparation(DNA模板文库制备）RandomPair-endImmobilizationofFragment

（DNA片段固定）Surface,BeadCovalentornon-covalentParallelSequencebySynthesis（平行测序反应）BaseextensionLigationSimultaneous

ImageAcquisition（光学图像采集与处理）FluorescenceChemiluminescenceSequenceReadandAssembly（序列拼接、组装）ClonalAmplification（DNA片段单分子扩增）EmulsionPCRPoloniesCycleWorkflowof2nd-GenSequencingSmallfragmentsAsymmetricAdaptorsIllumina/SolexaTechnologySmallfragmentsAsymmetricAdapSinglestrandedDNAfragmentspreparedandattachedtosolidsurface(单链片断固定到载片表面)DNAfragmentsareamplifiedtoformclusters（orpolonies）ofsingle-strandedtemplate(DNA簇群生成)Bridgeamplification

（桥式扩增法）Allpoloniesaresimultaneouslysequencedbypolymerizationandfluorescenceimagingcycles（循环合成反应+荧光成像）SolexaSequencingStepsSinglestrandedDNAfragmentsSmallfragments（DNA片段）AsymmetricAdaptors（不对称接头）

FragmentLibraryPrep（模板制备）LigateSmallfragmentsAsymmetricAdapAttachFragmentstoSurface

表面固定FlowCell载体芯片单链引物AttachFragmentstoSurface

表面BridgeFormation

“桥”的形成Anchoredprimersandtemplateswithinaradiuswillamplify.PrimaryampliconBridgeFormation

“桥”的形成AnchoreYieldtwocovalentmolecules3’3’BridgeAmplification

“桥”扩增Yieldtwocovalentmolecules3’Templatesbecomepermanentlybindtosurface.BridgeDenaturation

“桥”变性TemplatesbecomepermanentlyOne-moleculetemplateformsoneclusterOnlyonesetofprimersareused(B-orR-primer)经过30轮扩增，每个单分子得到了1000倍扩增，成为单克隆“DNA簇群”

Polonies

聚合酶族群生成One-moleculetemplate经过30轮扩增，SimultaneousSynthesis

同步链合成反应引物+DNA聚合酶+4种不同色荧光标记的可逆终止核苷酸SimultaneousSynthesis

同步链合成反应位于碱基3'末端的保护基团被除去，继续下一轮反应标记荧光经过扫描进行识别，读取该次反应颜色FluorescentImaging

荧光成像位于碱基3'末端的保护基团被除去，继续下一轮反应标记荧光经SecondSynthesis

第二轮合成SecondSynthesis

第二轮合成SecondImaging

第二轮成像SecondImaging

第二轮成像RepeatCycles

循环重复RepeatCycles

循环重复ProsThroughputisveryhigh–通量高Relativelyinexpensive–相对便宜ConsShortread–读长短ProsandCons（主要优缺点）ProsProsandCons（主要优缺点）Roche/454LifeSciencesGenomeSequencer试剂液体传送系统光学检测系统计算机系统Roche/454LifeSciencesGenomeSequencingMethodBasedonReal-timePyrophosphate（焦磷酸测序法）RonaghiM,UhlenM,NyrenPADepartmentofBiochemistryandBiotechnology,TheRoyalInstituteofTechnology,Stockholm,Sweden

Science1998;281:363,365SequencingMethodBasedonSinglestrandedDNAtemplatespreparedandattachedtosurfaceofmagneticbead(DNA单链片断固定在球珠表面)DNAfragmentisamplifiedthroughemPCR

(乳水包PCR扩增)

andenriched（筛选）Amplifiedbeadsaredepositedonflowcellwithmicrowells,onebeadperwell（球珠->微反应池）Eachbead,fixedinwell,issequencedbycyclingthrough

pyrophosphatechemistryandchemiluminescentimaging（循环合成反应+化学发光成像）Roche/454SequencingStepsSinglestrandedDNAtemplatesPyrophosphateChemistry

焦磷酸酶级联化学反应PPi+APS(adenosine5′phosphosulfate)ATPhydrolyzedbyluciferaseusingluciferintoproducelightRemove(d)NTPsandexcessATP由4种酶催化的同一反应体系中的化学发光反应(焦磷酸基团)(ATP硫酸化酶)

(荧光素酶)

(双磷酸酶)

BreakATPdown(聚合酶)（dATPS，dTTP，dCTP，dGTP四种核苷三磷酸）NPyrophosphateChemistry

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

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

（油包水）乳滴PCR扩增IVC:Invitrocompartmentalization将PCR反应物包被于“油包水”的乳化剂中，PCR扩增过程就可以在每一滴乳化剂内独立进行bead.

微型反应器

EmulsionPCR

（油包水）乳滴PCR扩增IVC:Roche/454WorkflowBeadDepositionOneamplifiedbeadpermicrowellFollowedbyenzymebeadsandpackingbeadsEnzymebeadsSulfurylaseLuciferasePackingbeadshelptokeepDNAbeadinmicrowellPyrosequencing4nucleotidessequentiallyflowinIncorporationofanucleotidereleasesapyrophosphate(PPi)SufurylaseconvertPPiintoATPATPhydrolyzedbyluciferaseusingluciferintoproducelightRoche/454WorkflowBeadDeposiRoche/454WorkflowImageAcquisitionCCDcameracoupledtothepicotiterplateChemiluminescentintensityreflectsnumberofnucleotideincorporatedineachflow;usedtodeterminehomopolymerregionUpto100cyclesrepeatedPost-acqProcessingDenovosequencingResequencingAmpliconvariantanalysisImageProcessingChemiluminescenteventmappedtowellFlowgramgeneratedforeachwellBasecalledRoche/454WorkflowImageAcquiProsThroughputishighrelativeto1st-gen–高通量Longerreadlength–高读长ConsReagentcosthigh–消耗试剂贵Difficultywithhomopolymerrun–难以分辨同聚物ProsandCons（主要优缺点）ProsProsandCons（主要优缺点）ABISOLiD–SequencebyLigationwithDual-baseEncoding

双基编码连接酶测序法ABISOLiD–SequencebyLigatiSinglestrandedDNAtemplatespreparedandattachedtobead(DNA单链片断固定在球珠表面)

DNAfragmentisamplifiedthroughemulsionPCR

(乳滴PCR扩增)

andenriched（筛选）Amplifiedbeadsattachedtoglassslidesurface（球珠被固定到玻璃载体表面）Eachbeadissequencedbycyclingthrough

ligationandfluorescenceimaging（循环连接反应+荧光成像）SOLiDSequencingSteps

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

4组荧光双碱基编码寡核苷酸探针Degeneratenucleotides简并碱基DinucleotidesFluorescentOligoOctamerProbSequencebyLigationHybridizationandligationofaspecificoligowhose1st&2ndbasesmatchthatofthetemplate连接寡核苷酸探针

SequencebyLigationHybridizatSequencebyLigationDetectionofthespecificfluorescence成像SequencebyLigationDetectionSequencebyLigation保护未连接链SequencebyLigation保护未连接链SequencebyLigationCleavageofallbasestothe5’ofbase5去除荧光标记SequencebyLigationCleavageoSequencebyLigation重复连接反应七次SequencebyLigation重复连接反应七次SequencebyLigationPrimerandallligatedportionsaremeltedfromthetemplateanddiscardedNewinitialprimerisusedthatisN-1inlength重启引物SequencebyLigationPrimerandSequencebyLigationGeneratesanoverlappingdataset重启后循环连接反应SequencebyLigationGeneratesFullSequenceCoverage

模板片段覆盖完毕FullSequenceCoverage

模板片段覆盖完Di-BaseEncodingColorSpace

双碱基编码颜色空间解读Di-BaseEncodingColorSpace

双SingleColorChange=SequencingErrorTwoColorChange=SNP(单核苷多态性）ColorSpaceErrorDetectionSingleColorChange=SequenciProsThroughputisveryhigh–高通量Build-inerrordetection–误差校正ConsShortreadlength–读长短ProsandCons（主要优缺点）ProsProsandCons（主要优缺点）模块化DNA分析系统的成功研制

（中科院北京基因组研究所/中科院半导体研究所）中国科学院重大科研装备研制项目2011年4月完成项目验收工作焦磷酸测序法+四工位平台达到国际主流测序设备技术指标：读长>500bp模块化DNA分析系统的成功研制

（中科院北京基因组研究所/中BIGIS-4第二代测序仪样机64BIGIS-系列已达国外主流设备性能指标BIGIS-4第二代测序仪样机64BIGIS-系列已达国外FluorogenicPyrosequencingin

PDMSMicroreactors

（荧光焦磷酸测序法）

FluorogenicPyrosequencinginLifeTechnologies’IonTorrentSequencingbysynthesissimilarto454(聚合酶合成测序法）Non-modifiednucleotidesflowinsequentiallyNon-lightbaseddetection（非光学检测）CMOSchipcontainH+sensitivewellsMonitorthereleaseofhydrogenionduringDNAsynthesiswith,essentially,tinypHmeters–通过跟踪在聚合成过程中氢离子的释放LifeTechnologies’IonTorrentSemiconductorSequencingIonProton™SequencerSemiconductorSequencingIonPIonTorrentCMOSChipIonTorrentCMOSChipIonTorrentSequencingIonTorrentSequencingProsCheaptobuild-无需光学检测Reagentcostlow

-无荧光标记物ConsLowthroughput

-密度有限Difficultywithhomopolymerrun–释放离子数难以分辨ProsandCons（主要优缺点）ProsProsandCons（主要优缺点）二代测序流程小结FragmentLibraryPreparation(DNA模板文库制备）RandomPair-endImmobilizationofFragment

（DNA片段固定）BeadFlatsurfaceParallelSequenceReaction（平行族群测序反应）PolymeraseLigaseSimultaneous

ImageAcquisition（图像采集与处理）FluorescenceChemiluminescenceSequenceReadandAssembly（序列拼接、组装）ClonalAmplification（DNA片段扩增）EmulsionPCRBridgeamplificationCycle二代测序流程小结FragmentLibraryPrepa二代测序平台比较SebastianJünemann,et.al.2013Volume31Number4NatureBiotechnology二代测序平台比较SebastianJünemann,et2nd-GenerationDrawbacks

二代测序平台的弱点Errorrateincreasewithlengthduetoensembleeffect（群体效应所导致的误差）Incompletechemicalreactions（化学反应效率不完全）Hindranceofpolymeraseincorporation（聚合反应缺陷）Deblocking(uncap)ofreversibleterminator（链终止解封反应）Opticalsignaldetectionerror（光信号检测误差）Strandsinanensemblenotinsync（失去族群同步性）De-phasinginanensemble->ambiguouslightsignal（族群光信号失相）Therefore,shortread-length（因而读序短）2nd-GenerationDrawbacks

二代测序平NextNext(3rd)-GenSequencing

SingleMoleculeSequencing(SMS)

三代单分子测序Sequencebysynthesis(SBS)insinglemolecule(单分子合成

)UsingfluorescentlylabelednucleotidesReal-timesinglemoleculefluorescencedetection(单分子荧光实时监测)Immobilizedpolymerasevs.immobilizedDNAtemplate（聚合酶固定vs.DNA模板固定）NextNext(3rd)-GenSequencingSMS单分子测序流程

FragmentLibraryPreparation(DNA模板文库制备）RandomPair-endImmobilizationofFragment

（DNA片段固定）FlatsurfaceParallelSequenceReaction（并行测序反应）PolymeraseSimultaneous

ImageAcquisition（图像采集与处理）FluorescenceSequenceReadandAssembly（序列拼接、组装）ClonalAmplification（DNA片段扩增）EmulsionPCRBridgeamplificationCycleSMS单分子测序流程FragmentLibraryPrKeyChallengesofSingleMoleculeMonitoring

SingleMolecule

Detection单分子监测Detectionoffluorescencefromsinglemoleculeinreal-time(对单分子荧光进行实时监测)

Interferenceofbackgroundfluorescence（背景荧光干扰）Non-specificbindingoffluorescentnucleotideonsurface（荧光标记核苷酸与表面的非特异性结合）Fluorescentlylabelednucleotidesinreactionsolution（漂浮在反应液的荧光标记物）

KeyChallengesofSingleMolecepifluorescentmicroscope

（落射荧光显微镜）mercurylampacooledCCDcameraSingleMoleculeFluorescenceMicroscope（荧光显微镜）

camera标本接物镜二色分光镜

epifluorescentmicroscope（落射荧Useevanescentwavetoilluminateandexcitefluorophoresinarestrictedregionadjacenttotheglass-waterinterface(用隐失波缩小荧光监测范围于玻水界面)Surfaceboundsinglemoleculescanbemonitoredwithhighsignaltonoise(固相表面结合的单分子荧光信噪比提高)TotalInternalReflectionFluorescence–TIR

全内反射荧光显微镜技术UseevanescentwavetoilluminTIRFSchematics

全内反射荧光显微镜工作原理显微镜载片

水介层

隐失波界面入射光

反射光

TIRFSchematics

全内反射荧光显微镜工作原理HeliScopeGeneticAnalyzer

tSMS–trueSingleMoleculeSequencingHeliScopeGeneticAnalyzer

tAcyclicprocessinvolvingmultipleroundsofA)incorporationoffluorescentnucleotide（核苷酸合成）

B)washing（核苷酸冲洗）

C)imaging（荧光成像）D)cleavinglabel（去除荧光标记）tSMSProcess单分子合成测序合成冲洗成像去除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（荧光成像系统）全内反射荧光显微镜ImagingSystem（荧光成像系统）全内反射荧光显微VisiGen(ABI)BiotechnologiesSequencebysynthesiswithfluorescenceresonanceenergytransfer(SBS-FRET)(荧光共振能量转移+合成测序)Polymerase+donorfluorophoreNucleotides+acceptorfluorophoreEnergytransferduringnucleotideincorporationAcceptoremitslightofaparticularwavelengthColoroflightusestoidentifythebaseVisiGen(ABI)BiotechnologiesSForster(Fluorescent)ResonanceEnergyTransfer(FRET)

荧光共振能量转移

Energytransfermechanismbetweentwofluorescentdyes(donorandacceptor)throughlongrangedipoledipoleinteractions(T.Förster1948)(荧光供体、受体间的能量转移）Onlyhappenwhenthedonorandacceptordyesareincloseproximity,<10nm,creatingaverysmallconfinementoftheacceptorexcitationlight

(近距能量转移）Forster(Fluorescent)Resonanc第二代测序技术——新一代基因组测序技术原理及应用课件OpticalDetectionSystem

光学探测系统光谱分离激发光源全内反射荧光OpticalDetectionSystem

光学探测系ZERO-MODEWAVEGUIDEs

零模波导

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

零模波导

forPacificBiosciences

ZeroModeWaveguide

零模波导PacificBiosciences’sSMRTchip

EachchipcontainsthousandsofZMWs.EachZMWisacylindricalholetensofnanometersindiameter,perforatingathinmetal(e.g.aluminum)filmsupportedbyatransparentsubstrate激发光源PacificBiosciences

ZeroMode

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

WhentheZMWisilluminateZMWwithAnchoredPolymeraseSelectiveimmobilizationofpolymerasetothefusedsilicaflooroftheZMWwasachievedbypassivationofthemetalcladdingsurfaceusingpolyphosphonatechemistry,producingenzymedensitycontrastsofglassoveraluminuminexcessof400:1.Yieldsofsingle-moleculeoccupanciesofapproximately30%wereobtainedforarangeofZMWdiameters.PolymeraseDNA固定聚合酶ZMWwithAnchoredPolymeraseSe核苷酸荧光标记Usefulinfocanbeobtainedfromsignalpulses核苷酸荧光标记Usefulinfocanbeobta物镜

色彩分离

激发出的荧光经光栅分光后打到单色CCD摄像机上同步成像荧光监测二色分光镜

物镜色彩分离荧光监测二色分光镜AdvantageofSMS

单分子测序优势

Noneedforamplification（不用扩增）Highinformationdensity（信息密度高）Theoreticallimitisdiffractionlimitoflight，λ/2（光衍射极限）Errorratestayflatvs.sequencelength（误差率不随链的延长增加）Longerreadlength（读序长）NodephasingissueduringsynthesisPotentialnewwayofdetectingmodifiednucleotide（提供修饰碱基检测新方法）AdvantageofSMS

单分子测序优势NoneChallengesforLight-basedSMS

光学单分子测序面对的问题SequencingAccuracy（精确性）Singlemoleculefluorescencedetection（单分子检测）Fluorophoreblinking（荧光间断）Polymerasefidelity（聚合酶保真度）SequencingReadLength（读长）Photophysicaldamage（光损害）Photo-bleachingtofluorophore（荧光团漂泊）DamagetoDNApolymerase（聚合酶损伤）ChallengesforLight-basedSMSFutureGeneration（4th-Gen）Physicalmethodandmeasurement（物理方法）Visualizingsequencingbymicrospectroscopy（基于电子显微镜直接测序法）Nano-structurebasedmeasurement

(基于纳米结构的测序法）Physicalandchemicalcombinedapproach（物理及化学相结合的手段）Stillininfancy（萌芽期）FutureGeneration（4th-Gen）Phys4th-GenerationBenefits

四代优势Labelfree（无标记物）+amplificationfree（无扩增）Noneedforopticalimagingsystem（无需光学显微镜）Simpledataprocessingandstorage（数据处理简易）Lowercost(低成本)+highspeed（高速度）4th-GenerationBenefits

四代优势LaMicroscopicSequencing（显微镜测序)

SimplyVisualizing

BasesUsingscanningprobemicroscopes（SPM,扫描探针显微镜）Atomicforcemicroscope(AFM，原子力显微镜

)CoupledwithRamanspectrumScanningtunnelingmicroscope(STM，扫描隧道显微镜)Direct-readGeneticSequencingbyZSGeneticswithElectronMicroscopeUsepolymerasewithheavyatomlabeled（重原子标记）nucleotidestosynthesisstrandtobedirectlyreadbyEMMicroscopicSequencing（显微镜测序)Solid-StateNanopore（固体纳米孔）

IonBeamSculpturedNanoporeinSiliconNitrideElectronBeamPunchedNanopore

onGrapheneSolid-StateNanopore（固体纳米孔）

IoBiologicalNanopore（生物纳米孔）

Nanoporewithengineeredprotein

(基因工程蛋白质改良纳米孔)α-hemolysin（溶血素蛋白）MspA（耻垢分枝杆菌孔蛋白）BiologicalNanopore（生物纳米孔）

NanKeyChallengesforNanopore-BasedSequencing

纳米孔测序的挑战DetectionofindividualbasesasDNApassingthrough(分辨不同碱基)ControloftraversingspeedofDNAchainthroughnanopore(DNA链穿孔速度控制)KeyChallengesforNanopore-BaNanoporeDetection

CurrentBlockade-阻塞电流NanoporeDetection

CurrentBloNanoporeDetection

ElectronTunneling–遂穿电流NanoporeDetection

ElectronTuDNA链纳米孔阻塞电流不同的碱基的阻塞电流不同DNA链纳米孔阻塞电流不同的碱基的阻塞电流不同NanoporeStrandSequencing聚合酶速度制控–生物分子马达NanoporeStrandSequencing聚合酶速NanoporeStrandSequencingNanoporeStrandSequencingApplications

应用

Applications

应用

ComparisonofNGSPlatforms

新一代测序平台比较ComparisonofNGSPlatforms

新一NGSApplicationTargets

新一代代测序平台应用对象Genomicanalysis(DNA)GenomeorDNAsequencingGenomicvariantdiscovery（基因组变异的发掘）GenomicalterationandmodificationTranscriptomicanalysis(RNA)TranscriptprofilingSmallRNAanalysisBiomarkerdiscoverybyProtona(Protein)Proof-of-conceptworkinsequencingprotein-boundoligonucleotideaptamer(蛋白/核酸适配体)NGSApplicationTargets

新一代代测序GenomeDenovosequencing（基因组初始测序）Initialgenerationoflargegenomesequence（基因组原始序列的产生）Productionofreferencegenomesequence(基因组数据库的建立）Wholegenomere-sequence(整基因组再测序）Discoveryofgenomicvariations（挖掘基因组变异）,e.g.SNP,indels,copynumber,rearrangement,etc.,amongpopulationUnderstandbiologicalconsequencesofvariantsTargetedre-sequence(定向再测序）SurveygenomicvariantsinregionsofinterestGenomeDenovosequencing（基因组初EukaryoticGenomeSequencingProjects（真核生物基因组）SubmittedtoNCBI;datatabulatedonMarch1,2010EukaryoticGenomeSequencingPDe-novoEukaryoticGenomeSequencingUsingNGSMostlydoneonSolexaand/or454platformDuetoshort-read,capillaryelectrophoresissequencersareoftenusedtofillthegap,e.g.repetitivesequenceDe-novoEukaryoticGenomeSequRe-sequencingofHumanGenomes

人类基因组再测序Aimtofindgenome-widevariationamongpopulations，e.g.SNP,indel,CN,….Byfar,themostcommonuseofNGSplatformsIlluminaGAhasbecomethedominantplatforminthisareaHighthroughputandrelativelylowruncosts454hastheadvantageoflongerreadbutconsumablecostishigherandhomopolymerissuesBioinformatictoolsforshortreadRe-sequencingofHumanGenomesRe-sequencingofHumanGenomesRe-sequencingofHumanGenomesTranscriptome转录组

WholeTranscriptomeShotgunSequencingorRNA-seqArevolutionarytoolfortranscriptomicsPriortothis,microarrayisthetoolofchoiceUtilizedeepsequencingcDNAwithNGStostudytranscriptsmRNAtranscriptanalysisDigitalgeneexpressionanalysis(数码基因差异表达）AlternativemRNAspliceforms(非传统RNA剪接）Discoveryandprofilingofgenome-widenon-codingsmallRNA（非编码RNA）ShortreadsareidealforstudyofnovelmiRNAandsiRNA(~30baselongwithregulatoryroles)Transcriptome转录组

WholeTransAdvantageofRNA-seqEliminatetheneedforclonalamplificationEmulsionPCRorbridgeamplificationNopriorknowledgeofgenomesequencerequiredMicroarrayrequiresitforconstructionofprobesDigitalcountingWiderdynamicrangeassequencinggetsdeeperRelativelylowcost(comparingwitholdtechniques)AdvantageofRNA-seqEliminateEpigenomics(表观基因组）Genome-wideDNA-proteinsinteraction（蛋白/核酸相互作用）Interactionoftranscriptionfactorwithitsdirecttargets（转录因子）Genomicprofilingofhistonemodification（组蛋白修饰）Genomicprofilingofnucleosomepositions（核小体定位）DNAmethylation（DNA甲基化）Epigenomics(表观基因组）Genome-wideChIP-seq

ChromatinImmunoprecipatation-DirectSequencing

染色质免疫共沉淀-测序Proteinofinterestcross-linkedwithDNATreatmentwithformaldehydeDNA-proteincomplexsheartogiverisesmallfragmentsUseantibodyspecifictotheproteintoenrichDNA-proteincomplexDNAreleasedfromproteinDNAfragmentssequencedbyNGSChIP-seq

ChromatinImmunoprecAdvantageofChIP-seq

(vs.ChIP-chip)Notlimitedbymicroarraycontent(不受限于微阵列内容）Don’tevenneedtohaveadraftgenomeassemblyavailableManyorganismscan,therefore,bestudiedNobiascausedbyprobehybridizationefficiency（无探针杂交偏爱性）Higherresolution（高分辨率）SinglebaseShortreadof35basessufficientAdvantageofChIP-seq

(vs.ChIMethylome(甲基化组)

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