新一代基因组测序技术原理及应用课件

NewGenerationsof

GenomicSequencingTechnologies&Applications新一代基因组测序技术原理及应用Outline

-

概要Sequencing

technology(技术）Historical

overview

（测序技术发展回顾

）Current

trends

in

sequencing

technology（测序技术的走向）2nd

Generation

Sequencer（第二代测序仪）NewNext-Generation-Singlemoleculesequencing

(SMDS)（第三代及单分子测序）Nano

and

direct

sequencing

(纳米及直接测序技术）Sequencing Applications

（应用）Conventionalsequencing

applicationsNewapplicationareasof

sequencingMethodsof

Sequencing测序主要方法Sequencing

by

separation（分离测序法）Sanger’schain-termination

methodMaxam&Gilbertchemical

methodSequencing

by

synthesis（合成测序法)Bybase

extensionBy

ligationSequencing

by

degradation（降解测序法)Sequencing

by

hybridization（杂交测序法)Oligo-probes

microarray（寡核苷酸探针微阵列芯片）+fluorescentlylabeledunknownDNA

fragmentsDirect

sequencing（直接测序法）Technological

Evolution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-terminator

sequencer（自动化荧光标记链终止测序法）2nd-Generation

(第二代，2006～Present)Sequencing

by

synthesis

in

essemble（DNA链合成测序法）3rd-Generation

(第三代，Present~3

years)Real-time

and

single

molecule

sequencing（实时、单分子合成测序法）4th-Generation

(第四代，in3~5

years?)Direct

sequencing（直接测序法）Related

Technologies与新一代测序有关的主要技术SouthernDNAhybridization

technique（DNA杂交技术）-

1975Sanger’s

chain-termination（链终止法）andMaxam、Gilbert’schemicalDNAsequencing

methods（化学裂解法）-

1977Automatedinsituoligonucleotidesynthesisinstrument（原位寡核苷酸合成仪）- 1980Mullis’s

discovery

of

PCR

at

Cetus

(聚合酶链式反应

DNA扩增)

–

1985Related

TechnologiesABI’s

automated

DNA

sequencer

(全自动核酸序列测定仪问世)

- 1986Affymatrix(Fodor’sgroup)first

gene-chip（原位合成基因芯片的制造）-

19922nd-generationDNAsequenceron

market（第二代测序仪问世）-

2006Single

molecule

sequencing（单分子测序技术涌现）-

2008～Bioinformatics（生物信息学）- midof

1980sDriverforSequencing

TechnologyPersonalGenome

Project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（测序发展动力）每一个人的基因组GenomicSequencingMoore’s

Law测序发展的摩尔定律APicture’s

Worth看图说话Now1Year2000(Celera

Genomics)400Complementarityof

Technologies各代间的兼容、互补性Technology技术Characters特征Applications

功能性PCR产物测序Denovo

新测序Re-seq

重测序GT1000/100WGSCBC1stGen第一代1.1Gel平板胶﹢﹢﹢﹢﹢NA1.2CE毛细管-四色荧光﹢﹢﹢﹢﹢﹢﹢﹢﹢﹢﹢NA2nd

Gen新一代2.1微乳液PCRLRNA﹢﹢﹢NA﹢﹢NANASRNA﹢﹢NA﹢﹢﹢NANA2.2高通量/无反应仓NA﹢﹢﹢NA﹢﹢﹢NANA2.3单分子NA﹢﹢﹢NA﹢﹢﹢﹢1000Future下一代3.1化学/纳米技术NA﹢﹢﹢NA﹢﹢﹢﹢10003.2纳米技术NA﹢﹢﹢NA﹢﹢﹢﹢1003.3纳米技术NA﹢﹢﹢NA﹢﹢﹢﹢100TrendsinNext-Gen

Sequencer新一代测序技术趋势Large-scale

and

high-throughput（大规模、高通量）Massively

parallel

processing（高度平行化）Microarray

of

clusters

or

beads

（固相微阵列）Simultaneous

sequencing

by

synthesis

(平行合成测序）Opticalmicroscopy

for

detection（光学检测)Component

miniaturization（装置微型化）Micro/nano

（微纳技术）Micro

fluidic（微观流体组件）Simplifiedprotocols（测序流程的简化）TeKey

Technology关键技术chnolog多学科交叉1st

Gen

第一代y

Conv增加并向微纳2nd

Gen

第二代ergenc技术倾斜3rd

Gen

第三代e4th

Gen

第四代SangerCyclic

ArraySMSDirectHybridization分子杂交√√√√Enzyme酶介导生化反应√√√Fluor-Label荧光标记底物√√√PCR扩增√√Electrophoresis电泳√Micro/Nano微纳加工√√√Fluor-Detection荧光信号识别√√√√SMD单分子信号检测√√Electro-Signal电信号识别√√Micro-Fluidics微量液流控制√√ConventionalSequencingTechnologies传统测序技术(byLadderFragments

Separation)Maxam、Gilbert’s

chemical

DNA

sequencingmethods

（化学裂解测序法）Sanger’sdideoxy-chainterminationmethod

(双脱氧链终止法)Dye-termination

method

(荧光标记链终止法）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Sequencingwith

FluorescentlyTagged

Chain-Terminator荧光标记链终止测序法1986LeroyHoodoftheCaliforniaInstituteofTechnologyandcolleaguesannouncethefirstautomatedDNAsequencingmachine1987AppliedBiosystemsInc.putsthefirstautomatedsequencingmachine,based

onHood'stechnology,onthemarketFour

fluorescentlylabeled

terminators（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FluorescenceSequencer荧光测序仪ABI

377

有64条泳道AutomatedDNASequencer

(CE)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Progressionof1st-GenerationThroughput第一代仪器测序通量演变1st–Generation

Limitation一代测序仪的局限Low

Throughput（通量低）Time-consumingseparationofchain-terminatedfragments（电泳分离耗时）Hardtoproducemassivelyparallelsystembasedelectrophoretic

separation（电泳分离不易大规模平行）High

Sequencing

Cost（成本高）Complexsample

preparation

&

handling（样品准备处理复杂）High

reagent

consumption（试剂用量大）Difficult

to

miniaturize（技术不易微量化）ShotgunSequencing鸟枪测序法DNAPolymerase合成测序法的基石

-

DNA聚合反应+ppi+

H+2nd-GenerationSequencingPlatforms二代测序平台Illumina/SolexaGenomic

AnalyzerSequencebysynthesiswithreversiblefluorescent

terminators（可逆性链终止合成测序）Roche/454LifeSciencesGS

FLXPyrosequencing（焦磷酸测序）ABI/AgencourtSOLiD

SystemSequencebyligationwithdualbaseencoding（双碱基编码连接测序）Workflowof2nd-Gen

Sequencing二代测序流程Fragment

LibraryPreparation(DNA模板文库制备）RandomPair-endImmobilization

ofFragment（DNA片段固定）Surface,

BeadCovalentor

non-covalentParallelSequence

bySynthesis（平行测序反应）Base

extensionLigationSimultaneousImageAcquisition（光学图像采集与处理）FluorescenceChemiluminescenceSequenceReadandAssembly（序列拼接、组装）Clonal

Amplification（DNA片段单分子扩增）Emulsion

PCRPoloniesCycleSmall

fragmentsIllumina/Solexa

TechnologySinglestrandedDNAfragmentspreparedandattachedtosolid

surface(单链片断固定到载片表面)DNAfragmentsareamplifiedtoformclusters（orpolonies）ofsingle-stranded

template

(DNA簇群生成)Bridge

amplification

（桥式扩增法）Allpoloniesaresimultaneouslysequencedbypolymerizationandfluorescence

imaging

cycles（循环合成反应+荧光成像）IlluminaSequencing

StepsSmall

fragments（DNA片段）AsymmetricAdaptors（不对称接头）Fragment

Library

Prep（模板制备）LigateAttachFragmentsto

Surface表面固定Flow

Cell载体芯片单链引物Bridge

Formation“桥”的形成Anchoredprimersandtemplateswithina

radiuswill

amplify.Primary

ampliconYieldtwocovalent

molecules3’3’Bridge

Amplification“桥”扩增Templatesbecome

permanentlybindto

surface.Bridge

Denaturation“桥”变性One-molecule

templateformsone

clusterOnlyonesetofprimersare

used(B-or

R-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Simultaneous

Synthesis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Fluorescent

Imaging荧光成像SecondCycleof

Synthesis第二轮合成Second

Imaging第二轮成像Repeat

Cycles循环重复ProsThroughput is

very

high

–

通量高Relatively

inexpensive

–

相对便宜ConsShort

read

–读长短Prosand

Cons（主要优缺点）Roche/454LifeSciencesGenome

Sequencer试剂液体传送系统光学检测系统计算机系统SequencingMethodBased

onReal-time

Pyrophosphate

（焦磷酸测序法）RonaghiM,UhlenM,Nyren

PADepartmentofBiochemistryandBiotechnology,TheRoyalInstituteofTechnology,Stockholm,SwedenScience1998;281:363,

365SinglestrandedDNAtemplatespreparedandattachedto

surfaceofmagnetic

bead

(DNA单链片断固定在球珠表面)DNA

fragment

is

amplified

through

emPCR

(乳水包PCR扩增)

and

enriched（筛选）Amplifiedbeadsaredepositedonflowcellwithmicrowells,

one

bead

per

well（球珠

->

微反应池）Eachbead,fixedinwell,issequencedbycyclingthroughpyrophosphatechemistryandchemiluminescent

imaging（循环合成反应

+化学发光成像）Roche/454Sequencing

StepsPyrophosphate

Chemistry焦磷酸酶级联化学反应PPi+APS

(adenosine5′

phosphosulfate)ATPhydrolyzedbyluciferaseusingluciferintoproducelightRemove(d)NTPs

andexcess

ATP(焦磷酸基团)(ATP硫酸化酶)(荧光素酶)(双磷酸酶)Break

ATPdown由4种酶催化的同一反应体系中的化学发光反应(聚合酶)（dATP

S，dTTP，dCTP，dGTP

四种核苷三磷酸）NRoche/454

Workflow（工作流程）DNA

LibraryPrepDNA

Fragmentend

repairedAsymmetricadaptors

ligatedDenatured->

sstemplateDNAlibraryEmulsion

PCR

Amplification

(乳滴PCR扩增）TemplateDNAimmobilizedonprimercoatedbeadsthruhybridization(1fragmentoneach

bead)Thermocycleto

amplifyAmplifiedbeadsenrichedwithstreptavidincoatedmagnetic

beadsEmulsion

PCR（油包水）乳滴PCR扩增ntalization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c行ompartbead.微型反应器Roche/454

WorkflowBead

DepositionOneamplifiedbeadper

microwellFollowedbyenzymebeadsandpackingbeadsEnzymebeadsSulfurylaseLuciferasePackingbeadshelptokeepDNAbeadin

microwellPyrosequencing4nucleotidessequentiallyflow

inIncorporationofanucleotidereleasesapyrophosphate

(PPi)SufurylaseconvertPPiinto

ATPATPhydrolyzedbyluciferaseusingluciferintoproduce

lightRoche/454

WorkflowImage

AcquisitionCCDcameracoupledtothe

picotiterplateChemiluminescentintensityreflectsnumberofnucleotideincorporatedineachflow;usedtodeterminehomopolymer

regionUpto100

cyclesrepeatedPost-acqProcessingDenovosequencingResequencingAmpliconvariant

analysisImageProcessingChemiluminescenteventmappedtowellFlowgramgeneratedforeachwellBase

calledProsThroughput

is

high

relative

to

1st-gen

–

高通量Longer

read

length

–

高读长ConsReagent

cost

high

–

消耗试剂贵Difficulty

with

homopolymerrun

–

难以分辨同聚物Prosand

Cons（主要优缺点）ABISOLiD–SequencebyLigationwithDual-base

Encoding双基编码连接酶测序法SinglestrandedDNAtemplatespreparedandattached

to

bead

(DNA单链片断固定在球珠表面)DNAfragmentisamplifiedthroughemulsionPCR

(乳滴PCR扩增)

and

enriched（筛选）Amplifiedbeadsattachedtoglassslide

surface（球珠被固定到玻璃载体表面）Eachbeadissequencedbycyclingthroughligation

and

fluorescence

imaging

（循环连接反应

+荧光成像）SOLiDSequencing

StepsABISOLiD–Library

PrepShearedfragmentsaretaggedwithadapters(A1andA2)toeachendABISOLiD–Emulsion

PCREmulsionPCRperformedusingDNAfragmentsfromlibraryonbeads(μm)

coatedwithoneofthe

primers3’-endofamplifiedDNAstrands

modifiedABISOLiD–Bead

DepositionAmplifiedbeadenrichedonpolystyrenecapturebeadscoatedwithA2adaptor;anybeadcontainingtheextendedproductswillbindpolystyrenebeadthroughitsP2end.ThisincreasethethroughputofbeadswithtargetedDNAfrom30%to

80%3’endofenrichedproductmodifiedtoallowcovalentattachmenttoglassslidesurface

randomlyFluorescentOligoOctamer

ProbesDegeneratenucleotides

简并碱基4组荧光双碱基编码寡核苷酸探针DinucleotidesSequenceby

LigationHybridizationandligationofaspecificoligowhose1st&2ndbasesmatchthatofthe

template连接寡核苷酸探针Sequenceby

LigationDetectionofthespecific

fluorescence成像Sequenceby

Ligation保护未连接链Sequenceby

Ligation去除荧光标记Cleavageofallbasestothe5’ofbase

5Sequenceby

Ligation重复连接反应七次Sequenceby

LigationPrimerandallligatedportionsaremeltedfromthetemplateand

discardedNewinitialprimerisusedthatisN-1in

length重启引物Sequenceby

LigationGeneratesanoverlappingdata

set重启后循环连接反应FullSequence

Coverage模板片段覆盖完毕Di-BaseEncodingColor

Space双碱基编码颜色空间解读Two

Color

Change

=

SNP

(单核苷多态性）ColorSpaceError

DetectionSingleColorChange=Sequencing

ErrorProsThroughput

is

very

high

–高通量Build-in

error

detection–

误差校正ConsShort

read

length

–读长短Prosand

Cons（主要优缺点）IonTorrentSequencingbysynthesissimilarto454(聚合酶合成测序法）Non-modifiednucleotidesflowin

sequentiallyNon-light

based

detection

（非光学检测）CMOSchipcontainH+sensitivewellsMonitorthereleaseofhydrogenionduringDNAsynthesiswith,essentially,

tiny

pH

meters

–通过跟踪在聚合成过程中氢离子的释放Semiconductor

Sequencing半导体芯片测序IonProton™

SequencerIon

Torrent

CMOS

芯片IonTorrent

SequencingProsCheap

to

build -

无需光学检测Reagent

cost

low -

无荧光标记物ConsLow

throughput -

密度有限Difficulty

with

homopolymerrun

–

释放离子数难以分辨Prosand

Cons

（主要优缺点）NGSMultiplexingwith

Barcode通过多标签进行多样本比对Summaryof2nd-Gen

Sequencing二代测序流程小结Fragment

LibraryPreparation(DNA模板文库制备）RandomPair-endImmobilization

ofFragment（DNA片段固定）BeadFlat

surfaceParallelSequenceReaction（平行族群测序反应）PolymeraseLigaseSimultaneousImageAcquisition（图像采集与处理）FluorescenceChemiluminescenceSequenceReadandAssembly（序列拼接、组装）ClonalAmplification（DNA片段扩增）Emulsion

PCRBridgeamplificationCycle2nd-GenPlatform

Comparison二代测序平台比较SebastianJünemann,et.al.2013Volume31Number4Nature

Biotechnology2nd-Generation

Drawbacks二代测序平台的弱点Errorrateincreasewithlengthduetoensemble

effect（群体效应所导致的误差）Incomplete

chemical

reactions（化学反应效率不完全）Hindrance

of

polymerase

incorporation

（聚合反应缺陷）Deblocking

(uncap)ofreversible

terminator（链终止解封反应）Optical

signal

detection

error（光信号检测误差）Strands

in

an

ensemble

not

in

sync

（失去族群同步性）De-phasing

in

an

ensemble

->

ambiguous

light

signal

（族群光信号失相）Therefore,

short

read-length（因而读序短）NextNext(3rd)-Gen

SequencingSingleMoleculeSequencing

(SMS)三代单分子测序Sequencebysynthesis(SBS)insinglemolecule

(单分子合成

)UsingfluorescentlylabelednucleotidesReal-timesinglemolecule

fluorescencedetection

(单分子荧光实时监测)Immobilizedpolymerasevs.immobilizedDNA

template（聚合酶固定

vs.

DNA模板固定）SMS单分子测序流程Fragment

LibraryPreparation(DNA模板文库制备）RandomPair-endImmobilization

ofFragment（DNA片段固定）Flat

surfaceParallelSequenceReaction（并行测序反应）PolymeraseSimultaneousImageAcquisition（图像采集与处理）FluorescenceSequenceReadandAssembly（序列拼接、组装）ClonalAmplification（DNA片段扩增）Emulsion

PCRBridgeamplificationCycleKeyChallengesofSingleMolecule

MonitoringSingle

Molecule

Detection

单分子监测Detectionoffluorescencefromsinglemoleculein

real-time

(对单分子荧光进行实时监测)Interference

of

background

fluorescence（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）epifluorescent

microscope（落射荧光显微镜）mercury

lampacooledCCD

cameraSingleMolecule

FluorescenceMicroscope（荧光显微镜）camera标本接物镜二色分光镜TotalInternalReflectionFluorescence–

TIR全内反射荧光显微镜技术Useevanescentwavetoilluminateandexcitefluorophoresinarestrictedregionadjacenttotheglass-water

interface

(用隐失波缩小荧光监测范围于玻水界面)Surfaceboundsinglemoleculescanbemonitored

with

high

signal

to

noise

(固相表面结合的单分子荧光信噪比提高)TIRF

Schematics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HeliScopeGenetic

AnalyzertSMS–trueSingleMolecule

SequencingA)incorporationoffluorescent

nucleotide（核苷酸合成）B)

washing（核苷酸冲洗）C)

imaging（荧光成像）D)

cleaving

label（去除荧光标记）tSMS

Process单分子合成测序Acyclicprocessinvolvingmultiplerounds

of合成冲洗成像去除VisiGen(ABI)

BiotechnologiesSequencebysynthesiswithfluorescenceresonanceenergytransfer(SBS-FRET)

(荧光共振能量转移+合成测序)Polymerase+donor

fluorophoreNucleotides+acceptor

fluorophoreEnergytransferduringnucleotide

incorporationAcceptoremitslightofaparticular

wavelengthColoroflightusestoidentifythe

baseForster(Fluorescent)ResonanceEnergyTransfer

(FRET)荧光共振能量转移Energytransfermechanismbetweentwofluorescentdyes(donorandacceptor)throughlongrangedipoledipoleinteractions(T.

Förster

1948)

(荧光供体、受体间的能量转移）Onlyhappenwhenthedonorandacceptordyesareincloseproximity,<10nm,creatingaverysmallconfinementoftheacceptor

excitation

light

(近距能量转移）OpticalDetection

System光学探测系统光谱分离全内反射荧光激发光源3rd-Gen:Pacific

BiosciencesPacBioRS

IIWorkflow

工作流程度长、精确度ZERO-MODE

WAVEGUIDEs零模波导forSingle-MoleculeAnalysisatHigh

ConcentrationsM.J.Levene,J.Korlach,S.W.Turner,M.Foquet,H.G.Craighead,W.W.WebbCornellUniversity,ClarkHall,Ithaca,NY14853,

USAOpticalapproachesforobservingthedynamicsofsinglemoleculeshaverequiredpico-tonanomolarconcentrationsoffluorophoreinordertoisolateindividualmolecules.However,manybiologicallyrelevantprocessesoccuratmicromolarligandconcentrations,necessitatingareductionintheconventionalobservationvolumebythreeordersofmagnitude.WeshowthatarraysofZERO-MODEWAVEGUIDEsconsistingofsubwavelengthholesinametalfilmprovideasimpleandhighlyparallelmeansforstudyingsingle-moleculedynamicsatmicromolarconcentrationswithmicrosecondtemporalresolution.WepresentobservationsofDNApolymeraseactivityasanexampleoftheeffectivenessofZERO-MODEWAVEGUIDEsforperformingsingle-moleculeexperimentsathigh

concentrations.Science31January2003:

682ZeroMode

Waveguide零模波导

PacificBiosciences’sSMRTchip

EachchipcontainsthousandsofZMWs.EachZMWisacylindricalhole

tensofnanometersindiameter,perforatingathinmetal(e.g.aluminum)filmsupportedbyatransparent

substrate激发光源WhentheZMWisilluminatedthroughthetransparentsubstratebylaserlight,thewavelengthofthelightistoolargeto

passthroughthewaveguide’saperture.Attenuatedevanescentlightfrom

theexcitationbeamdecaysexponentiallyandpenetratesthelower20-30nmofeachwaveguide,creatingadetectionvolumeofonly20zeptoliters(10-21liters).Thisdramaticreductioninthedetectionvolumeprovidestheneeded1000-foldimprovementinrejectionofbackground

fluorescence.隐失波Ø<<λ隐失波ZMWwithAnchored

Polymerase零模波导

+

固定DNA聚合酶SelectiveimmobilizationofpolymerasetothefusedsilicaflooroftheZMWwasachievedbypassivationofthemetalcladdingsurfaceusingpolyphosphonatechemistry,producingenzymedensitycontrastsofglassoveraluminuminexcessof400:1.Yieldsofsingle-molecule

occupanciesofapproximately30%wereobtainedforarangeofZMW

diameters.PolymeraseDNA固定聚合酶核苷酸荧光标记Usefulinfocanbeobtainedfromsignal

pulses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PacBioRSIIAdvantagesLongreads:averagelengthsof4,200to

8,500Highaccuracy:with99%

accuracyDetectDNAmodifications:5-methylcytosine,N6-methyladenine,N4-methylcytosine,oxidative

damage.LeastGC

bias.Noamplification:noPCR

required.Advantageof

SMS单分子测序优势No

need

for

amplification（不用扩增）High

information

density（信息密度高）Theoretical

limit

is

diffraction

limit

of

light，λ/2（光衍射极限）Error

rate

stay

flatvs.sequence

length（误差率不随链的延长增加）Longer

readlength（读序长）Nodephasingissueduring

synthesisPotentialnewwayofdetectingmodified

nucleotide（提供修饰碱基检测新方法）ChallengesforLight-based

SMS光学单分子测序面对的问题Sequencing

Accuracy

（精确性）Single

molecule

fluorescence

detection

（单分子检测）Fluorophore

blinking

（荧光间断）Polymerase

fidelity

（聚合酶保真度）Sequencing

ReadLength

（读长）Photophysical

damage

（光损害）Photo-bleaching

to

fluorophore

（荧光团漂泊）Damage

to

DNA

polymerase

（聚合酶损伤）Future

Generation（4th-Gen）Physical

method

andmeasurement（物理方法）Visualizingsequencingby

microspectroscopy（基于电子显微镜直接测序法）Nano-structurebased

measurement

(基于纳米结构的测序法）Physicalandchemicalcombinedapproach（物理及化学相结合的手段）Still

in

infancy

（萌芽期）4th-Generation

Benefits四代优势Label

free（无标记物）+amplification

free（无扩增）No

need

for

optical

imaging

system（无需光学显微镜）Simple

data

processing

and

storage（数据处理简易）Lower

cost

(低成本)

+

high

speed（高速度）Microscopic

Sequencing（显微镜测序)SimplyVisualizing

BasesUsingscanningprobemicroscopes（SPM,

扫描探针显微镜）Atomic

force

microscope(AFM，原子力显微镜

)CoupledwithRamanspectrumScanning

tunneling

microscope

(STM，扫描隧道显微镜)Direct-readGeneticSequencingbyZSGeneticswithElectron

MicroscopeUse

polymerase

with

heavy

atom

labeled

（重原子标记）nucleotidestosynthesisstrandtobedirectlyreadbyEMSolid-State

Nanopore（固体纳米孔）IonBeam

SculpturedNanoporeinSiliconNitrideElectronBeamPunchedNanoporeonGrapheneBiological

Nanopore（生物纳米孔）Nanopore

with

engineered

protein

(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）KeyChallengesforNanopore-Based

Sequencing纳米孔测序的挑战DetectionofindividualbasesasDNA

passing

through

(分辨不同碱基)ControloftraversingspeedofDNAchain

through

nanopore

(DNA链穿孔速度控制)NanoporeDetectionCurrent

Blockade

-

阻塞电流DNA链纳米孔阻塞电流不同的碱基的阻塞电流不同速度控制Polymerase 聚合酶Speedcontrolthrumolecularmotor速度制控–

生物分子马达NanoporeStrand

SequencingApplications应用ComparisonofNGSPlatforms新一代测序平台比较PlatformInstrumentCostAmplificationGb/RunReadLengthRun

TimeCost/MbRoche

454$500,00Emulsion

PCR0.14009

hrs$84SOLiD$595,000Emulsion

PCR335-505

days$6Illumina

GA$540,000BridgeAmplification1.335-1004

days$7Helicos$900,000N/A37328

days$0.5Pacific

Bio$695,000N/A3~1100(upto

5000)12

hrs?NGSApplication

Targets新一代代测序平台应用对象Genomicanalysis

(DNA)GenomeorDNAsequencingGenomic

variant

discovery

（基因组变异的发掘）Genomicalterationand

modificationTranscriptomicanalysis(RNA)Transcript

profilingSmallRNA

analysisBiomarkerdiscoverybyProtona

(Protein)Proof-of-conceptworkinsequencingprotein-bound

oligonucleotide

aptamer

(蛋白/核酸适配体)EukaryoticGenomeSequencingProjects

（真核生物基因组）OrganismGroupCompletedDraftAssemblyIn

ProgressSUMAnimals4137146287Plants32385111Fungi1012093223Protists64964119TOTAL23329388740SubmittedtoNCBI;datatabulatedonMarch1,

2010De-novoEukaryoticGenomeSequencingUsing

NGSOrganismGroupStatusGenomeSize

(Mb)Platform(s)DepthAiluropodamelanoleucaAnimalsAssembly2460Solexa56xGrosmanniaclavigeraFungiAssembly32.5Solexa,454,and

Sanger50xVitis

viniferaPlantsAssembly500454

andSanger11xCucumissativusPlantsAssembly367Solexa

andSanger72xGiant

PandaAnimalsAssembly2250Solexa73xMostlydoneonSolexaand/or454

platformDuetoshort-read,capillaryelectrophoresissequencers

areoftenusedtofillthegap,e.g.repetitive

sequenceRe-sequencingofHuman

Genomes人类基因组再测序Aimtofindgenome-widevariationamongpopulations，e.g.SNP,indel,CN,

….Byfar,themostcommonuseofNGS

platformsIlluminaGAhasbecomethedominantplatforminthis

areaHighthroughputandrelativelylowrun

costs454hastheadvantageoflongerreadbutconsumablecostishigherandhomopolymerissuesBioinformatictoolsforshort

readRe-sequencingofHuman

GenomesPlatformIndividualNo.ofRead

(Mb)ReadLengthReadCoverageNo.

ofRunsEst.Cost(in

1000$)SangerJ.C.

Venter328007.5x>340,00070,000Roche

454J.

Watson932507.4x2341,000SOLiDJ.

Lupski2383530x375SolexaAn

AfricanMale3,6813540x40250An

HanChinese2,9503536x354001st

KoreanMale1,64735,7429x152502nd

KoreanMale1,91036,88,10628x30200HelicosS.

Quake2,7253228x448Transcriptome

转录组WholeTranscriptomeShotgunSequencingorRNA-seqArevolutionarytoolfor

transcriptomicsPriortothis,microarrayisthetoolof

choiceUtilize

deep

sequencing cDNAwithNGSto

studytranscriptsmRNAtranscript

analysisDigital

gene

expression analysis(数码基因差异表达）Alternative

mRNA

splice

forms

(非传统RNA剪接）Discoveryandprofilingofgenome-widenon-coding

small

RNA

（非编码RNA）ShortreadsareidealforstudyofnovelmiRNAandsiRNA(~30baselongwithregulatory

roles)Advantageof

RNA-seqEliminatetheneedforclonal

amplificationEmulsionPCRorbridge

amplificationNopriorknowledgeofgenome

sequencerequiredMicroarrayrequiresitforconstructionof

probesDigital

countingWiderdynamicrangeassequencinggets

deeperRelativelylowcost(comparingwitholdtechniques)Epigenomics(表观基因组）Genome-wide

DNA-proteins

interaction（蛋白/核酸相互作用）Interactionoftranscriptionfactorwithitsdirecttargets

（转录因子）Genomic

profiling

of

histone

modification

（组蛋白修饰）Genomic

profiling

of

nucleosome

positions

（核小体定位）DNA

methylation

（DNA甲基化）ChIP-seqChromatinImmunoprecipatation-DirectSequencing染色质免疫共沉淀-测序Proteinofinterestcross-linkedwith

DNATreatmentwith

formaldehydeDNA-proteincomplexsheartogiverisesmall

fr