新一代基因組測(cè)序技術(shù)原理及應(yīng)用課件

NewGenerationsof

GenomicSequencingTechnologies&Applications新一代基因組測(cè)序技術(shù)原理及應(yīng)用Outline

-

概要Sequencing

technology(技術(shù)）Historical

overview

（測(cè)序技術(shù)發(fā)展回顧

）Current

trends

in

sequencing

technology（測(cè)序技術(shù)的走向）2nd

Generation

Sequencer（第二代測(cè)序儀）NewNext-Generation-Singlemoleculesequencing

(SMDS)（第三代及單分子測(cè)序）Nano

and

direct

sequencing

(納米及直接測(cè)序技術(shù)）Sequencing Applications

（應(yīng)用）Conventionalsequencing

applicationsNewapplicationareasof

sequencingMethodsof

Sequencing測(cè)序主要方法Sequencing

by

separation（分離測(cè)序法）Sanger’schain-termination

methodMaxam&Gilbertchemical

methodSequencing

by

synthesis（合成測(cè)序法)Bybase

extensionBy

ligationSequencing

by

degradation（降解測(cè)序法)Sequencing

by

hybridization（雜交測(cè)序法)Oligo-probes

microarray（寡核苷酸探針微陣列芯片）+fluorescentlylabeledunknownDNA

fragmentsDirect

sequencing（直接測(cè)序法）Technological

Evolution測(cè)序發(fā)展過(guò)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-terminator

sequencer（自動(dòng)化熒光標(biāo)記鏈終止測(cè)序法）2nd-Generation

(第二代，2006～Present)Sequencing

by

synthesis

in

essemble（DNA鏈合成測(cè)序法）3rd-Generation

(第三代，Present~3

years)Real-time

and

single

molecule

sequencing（實(shí)時(shí)、單分子合成測(cè)序法）4th-Generation

(第四代，in3~5

years?)Direct

sequencing（直接測(cè)序法）Related

Technologies與新一代測(cè)序有關(guān)的主要技術(shù)SouthernDNAhybridization

technique（DNA雜交技術(shù)）-

1975Sanger’s

chain-termination（鏈終止法）andMaxam、Gilbert’schemicalDNAsequencing

methods（化學(xué)裂解法）-

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

discovery

of

PCR

at

Cetus

(聚合酶鏈?zhǔn)椒磻?yīng)

DNA擴(kuò)增)

–

1985Related

TechnologiesABI’s

automated

DNA

sequencer

(全自動(dòng)核酸序列測(cè)定儀問(wèn)世)

- 1986Affymatrix(Fodor’sgroup)first

gene-chip（原位合成基因芯片的制造）-

19922nd-generationDNAsequenceron

market（第二代測(cè)序儀問(wèn)世）-

2006Single

molecule

sequencing（單分子測(cè)序技術(shù)涌現(xiàn)）-

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

1980sDriverforSequencing

TechnologyPersonalGenome

Project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(lái)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（測(cè)序發(fā)展動(dòng)力）每一個(gè)人的基因組GenomicSequencingMoore’s

Law測(cè)序發(fā)展的摩爾定律APicture’s

Worth看圖說(shuō)話Now1Year2000(Celera

Genomics)400Complementarityof

Technologies各代間的兼容、互補(bǔ)性Technology技術(shù)Characters特征Applications

功能性PCR產(chǎn)物測(cè)序Denovo

新測(cè)序Re-seq

重測(cè)序GT1000/100WGSCBC1stGen第一代1.1Gel平板膠﹢﹢﹢﹢﹢NA1.2CE毛細(xì)管-四色熒光﹢﹢﹢﹢﹢﹢﹢﹢﹢﹢﹢NA2nd

Gen新一代2.1微乳液PCRLRNA﹢﹢﹢NA﹢﹢NANASRNA﹢﹢NA﹢﹢﹢NANA2.2高通量/無(wú)反應(yīng)倉(cāng)NA﹢﹢﹢NA﹢﹢﹢NANA2.3單分子NA﹢﹢﹢NA﹢﹢﹢﹢1000Future下一代3.1化學(xué)/納米技術(shù)NA﹢﹢﹢NA﹢﹢﹢﹢10003.2納米技術(shù)NA﹢﹢﹢NA﹢﹢﹢﹢1003.3納米技術(shù)NA﹢﹢﹢NA﹢﹢﹢﹢100TrendsinNext-Gen

Sequencer新一代測(cè)序技術(shù)趨勢(shì)Large-scale

and

high-throughput（大規(guī)模、高通量）Massively

parallel

processing（高度平行化）Microarray

of

clusters

or

beads

（固相微陣列）Simultaneous

sequencing

by

synthesis

(平行合成測(cè)序）Opticalmicroscopy

for

detection（光學(xué)檢測(cè))Component

miniaturization（裝置微型化）Micro/nano

（微納技術(shù)）Micro

fluidic（微觀流體組件）Simplifiedprotocols（測(cè)序流程的簡(jiǎn)化）TeKey

Technology關(guān)鍵技術(shù)chnolog多學(xué)科交叉1st

Gen

第一代y

Conv增加并向微納2nd

Gen

第二代ergenc技術(shù)傾斜3rd

Gen

第三代e4th

Gen

第四代SangerCyclic

ArraySMSDirectHybridization分子雜交√√√√Enzyme酶介導(dǎo)生化反應(yīng)√√√Fluor-Label熒光標(biāo)記底物√√√PCR擴(kuò)增√√Electrophoresis電泳√Micro/Nano微納加工√√√Fluor-Detection熒光信號(hào)識(shí)別√√√√SMD單分子信號(hào)檢測(cè)√√Electro-Signal電信號(hào)識(shí)別√√Micro-Fluidics微量液流控制√√ConventionalSequencingTechnologies傳統(tǒng)測(cè)序技術(shù)(byLadderFragments

Separation)Maxam、Gilbert’s

chemical

DNA

sequencingmethods

（化學(xué)裂解測(cè)序法）Sanger’sdideoxy-chainterminationmethod

(雙脫氧鏈終止法)Dye-termination

method

(熒光標(biāo)記鏈終止法）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)Sequencingwith

FluorescentlyTagged

Chain-Terminator熒光標(biāo)記鏈終止測(cè)序法1986LeroyHoodoftheCaliforniaInstituteofTechnologyandcolleaguesannouncethefirstautomatedDNAsequencingmachine1987AppliedBiosystemsInc.putsthefirstautomatedsequencingmachine,based

onHood'stechnology,onthemarketFour

fluorescentlylabeled

terminators（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(kāi)，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FluorescenceSequencer熒光測(cè)序儀ABI

377

有64條泳道AutomatedDNASequencer

(CE)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Progressionof1st-GenerationThroughput第一代儀器測(cè)序通量演變1st–Generation

Limitation一代測(cè)序儀的局限Low

Throughput（通量低）Time-consumingseparationofchain-terminatedfragments（電泳分離耗時(shí)）Hardtoproducemassivelyparallelsystembasedelectrophoretic

separation（電泳分離不易大規(guī)模平行）High

Sequencing

Cost（成本高）Complexsample

preparation

&

handling（樣品準(zhǔn)備處理復(fù)雜）High

reagent

consumption（試劑用量大）Difficult

to

miniaturize（技術(shù)不易微量化）ShotgunSequencing鳥(niǎo)槍測(cè)序法DNAPolymerase合成測(cè)序法的基石

-

DNA聚合反應(yīng)+ppi+

H+2nd-GenerationSequencingPlatforms二代測(cè)序平臺(tái)Illumina/SolexaGenomic

AnalyzerSequencebysynthesiswithreversiblefluorescent

terminators（可逆性鏈終止合成測(cè)序）Roche/454LifeSciencesGS

FLXPyrosequencing（焦磷酸測(cè)序）ABI/AgencourtSOLiD

SystemSequencebyligationwithdualbaseencoding（雙堿基編碼連接測(cè)序）Workflowof2nd-Gen

Sequencing二代測(cè)序流程Fragment

LibraryPreparation(DNA模板文庫(kù)制備）RandomPair-endImmobilization

ofFragment（DNA片段固定）Surface,

BeadCovalentor

non-covalentParallelSequence

bySynthesis（平行測(cè)序反應(yīng)）Base

extensionLigationSimultaneousImageAcquisition（光學(xué)圖像采集與處理）FluorescenceChemiluminescenceSequenceReadandAssembly（序列拼接、組裝）Clonal

Amplification（DNA片段單分子擴(kuò)增）Emulsion

PCRPoloniesCycleSmall

fragmentsIllumina/Solexa

TechnologySinglestrandedDNAfragmentspreparedandattachedtosolid

surface(單鏈片斷固定到載片表面)DNAfragmentsareamplifiedtoformclusters（orpolonies）ofsingle-stranded

template

(DNA簇群生成)Bridge

amplification

（橋式擴(kuò)增法）Allpoloniesaresimultaneouslysequencedbypolymerizationandfluorescence

imaging

cycles（循環(huán)合成反應(yīng)+熒光成像）IlluminaSequencing

StepsSmall

fragments（DNA片段）AsymmetricAdaptors（不對(duì)稱接頭）Fragment

Library

Prep（模板制備）LigateAttachFragmentsto

Surface表面固定Flow

Cell載體芯片單鏈引物Bridge

Formation“橋”的形成Anchoredprimersandtemplateswithina

radiuswill

amplify.Primary

ampliconYieldtwocovalent

molecules3’3’Bridge

Amplification“橋”擴(kuò)增Templatesbecome

permanentlybindto

surface.Bridge

Denaturation“橋”變性O(shè)ne-molecule

templateformsone

clusterOnlyonesetofprimersare

used(B-or

R-primer)經(jīng)過(guò)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Simultaneous

Synthesis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(guò)掃描進(jìn)行識(shí)別，讀取該次反應(yīng)顏色Fluorescent

Imaging熒光成像SecondCycleof

Synthesis第二輪合成Second

Imaging第二輪成像Repeat

Cycles循環(huán)重復(fù)ProsThroughput is

very

high

–

通量高Relatively

inexpensive

–

相對(duì)便宜ConsShort

read

–讀長(zhǎng)短Prosand

Cons（主要優(yōu)缺點(diǎn)）Roche/454LifeSciencesGenome

Sequencer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)SequencingMethodBased

onReal-time

Pyrophosphate

（焦磷酸測(cè)序法）RonaghiM,UhlenM,Nyren

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

365SinglestrandedDNAtemplatespreparedandattachedto

surfaceofmagnetic

bead

(DNA單鏈片斷固定在球珠表面)DNA

fragment

is

amplified

through

emPCR

(乳水包PCR擴(kuò)增)

and

enriched（篩選）Amplifiedbeadsaredepositedonflowcellwithmicrowells,

one

bead

per

well（球珠

->

微反應(yīng)池）Eachbead,fixedinwell,issequencedbycyclingthroughpyrophosphatechemistryandchemiluminescent

imaging（循環(huán)合成反應(yīng)

+化學(xué)發(fā)光成像）Roche/454Sequencing

StepsPyrophosphate

Chemistry焦磷酸酶級(jí)聯(lián)化學(xué)反應(yīng)PPi+APS

(adenosine5′

phosphosulfate)ATPhydrolyzedbyluciferaseusingluciferintoproducelightRemove(d)NTPs

andexcess

ATP(焦磷酸基團(tuán))(ATP硫酸化酶)(熒光素酶)(雙磷酸酶)Break

ATPdown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)(聚合酶)（dATP

S，dTTP，dCTP，dGTP

四種核苷三磷酸）NRoche/454

Workflow（工作流程）DNA

LibraryPrepDNA

Fragmentend

repairedAsymmetricadaptors

ligatedDenatured->

sstemplateDNAlibraryEmulsion

PCR

Amplification

(乳滴PCR擴(kuò)增）TemplateDNAimmobilizedonprimercoatedbeadsthruhybridization(1fragmentoneach

bead)Thermocycleto

amplifyAmplifiedbeadsenrichedwithstreptavidincoatedmagnetic

beadsEmulsion

PCR（油包水）乳滴PCR擴(kuò)增ntalization將PCR反應(yīng)物包被于“油包水”的乳化劑中，PCR擴(kuò)增過(guò)程就可以在每一滴乳化劑內(nèi)獨(dú)立進(jìn)c行ompartbead.微型反應(yīng)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

–

高讀長(zhǎng)ConsReagent

cost

high

–

消耗試劑貴Difficulty

with

homopolymerrun

–

難以分辨同聚物Prosand

Cons（主要優(yōu)缺點(diǎn)）ABISOLiD–SequencebyLigationwithDual-base

Encoding雙基編碼連接酶測(cè)序法SinglestrandedDNAtemplatespreparedandattached

to

bead

(DNA單鏈片斷固定在球珠表面)DNAfragmentisamplifiedthroughemulsionPCR

(乳滴PCR擴(kuò)增)

and

enriched（篩選）Amplifiedbeadsattachedtoglassslide

surface（球珠被固定到玻璃載體表面）Eachbeadissequencedbycyclingthroughligation

and

fluorescence

imaging

（循環(huán)連接反應(yīng)

+熒光成像）SOLiDSequencing

StepsABISOLiD–Library

PrepShearedfragmentsaretaggedwithadapters(A1andA2)toeachendABISOLiD–Emulsion

PCREmulsionPCRperformedusingDNAfragmentsfromlibraryonbeads(μm)

coatedwithoneofthe

primers3’-endofamplifiedDNAstrands

modifiedABISOLiD–Bead

DepositionAmplifiedbeadenrichedonpolystyrenecapturebeadscoatedwithA2adaptor;anybeadcontainingtheextendedproductswillbindpolystyrenebeadthroughitsP2end.ThisincreasethethroughputofbeadswithtargetedDNAfrom30%to

80%3’endofenrichedproductmodifiedtoallowcovalentattachmenttoglassslidesurface

randomlyFluorescentOligoOctamer

ProbesDegeneratenucleotides

簡(jiǎn)并堿基4組熒光雙堿基編碼寡核苷酸探針DinucleotidesSequenceby

LigationHybridizationandligationofaspecificoligowhose1st&2ndbasesmatchthatofthe

template連接寡核苷酸探針Sequenceby

LigationDetectionofthespecific

fluorescence成像Sequenceby

Ligation保護(hù)未連接鏈Sequenceby

Ligation去除熒光標(biāo)記Cleavageofallbasestothe5’ofbase

5Sequenceby

Ligation重復(fù)連接反應(yīng)七次Sequenceby

LigationPrimerandallligatedportionsaremeltedfromthetemplateand

discardedNewinitialprimerisusedthatisN-1in

length重啟引物Sequenceby

LigationGeneratesanoverlappingdata

set重啟后循環(huán)連接反應(yīng)FullSequence

Coverage模板片段覆蓋完畢Di-BaseEncodingColor

Space雙堿基編碼顏色空間解讀Two

Color

Change

=

SNP

(單核苷多態(tài)性）ColorSpaceError

DetectionSingleColorChange=Sequencing

ErrorProsThroughput

is

very

high

–高通量Build-in

error

detection–

誤差校正ConsShort

read

length

–讀長(zhǎng)短Prosand

Cons（主要優(yōu)缺點(diǎn)）IonTorrentSequencingbysynthesissimilarto454(聚合酶合成測(cè)序法）Non-modifiednucleotidesflowin

sequentiallyNon-light

based

detection

（非光學(xué)檢測(cè)）CMOSchipcontainH+sensitivewellsMonitorthereleaseofhydrogenionduringDNAsynthesiswith,essentially,

tiny

pH

meters

–通過(guò)跟蹤在聚合成過(guò)程中氫離子的釋放Semiconductor

Sequencing半導(dǎo)體芯片測(cè)序IonProton?

SequencerIon

Torrent

CMOS

芯片IonTorrent

SequencingProsCheap

to

build -

無(wú)需光學(xué)檢測(cè)Reagent

cost

low -

無(wú)熒光標(biāo)記物ConsLow

throughput -

密度有限D(zhuǎn)ifficulty

with

homopolymerrun

–

釋放離子數(shù)難以分辨Prosand

Cons

（主要優(yōu)缺點(diǎn)）NGSMultiplexingwith

Barcode通過(guò)多標(biāo)簽進(jìn)行多樣本比對(duì)Summaryof2nd-Gen

Sequencing二代測(cè)序流程小結(jié)Fragment

LibraryPreparation(DNA模板文庫(kù)制備）RandomPair-endImmobilization

ofFragment（DNA片段固定）BeadFlat

surfaceParallelSequenceReaction（平行族群測(cè)序反應(yīng)）PolymeraseLigaseSimultaneousImageAcquisition（圖像采集與處理）FluorescenceChemiluminescenceSequenceReadandAssembly（序列拼接、組裝）ClonalAmplification（DNA片段擴(kuò)增）Emulsion

PCRBridgeamplificationCycle2nd-GenPlatform

Comparison二代測(cè)序平臺(tái)比較SebastianJünemann,et.al.2013Volume31Number4Nature

Biotechnology2nd-Generation

Drawbacks二代測(cè)序平臺(tái)的弱點(diǎn)Errorrateincreasewithlengthduetoensemble

effect（群體效應(yīng)所導(dǎo)致的誤差）Incomplete

chemical

reactions（化學(xué)反應(yīng)效率不完全）Hindrance

of

polymerase

incorporation

（聚合反應(yīng)缺陷）Deblocking

(uncap)ofreversible

terminator（鏈終止解封反應(yīng)）Optical

signal

detection

error（光信號(hào)檢測(cè)誤差）Strands

in

an

ensemble

not

in

sync

（失去族群同步性）De-phasing

in

an

ensemble

->

ambiguous

light

signal

（族群光信號(hào)失相）Therefore,

short

read-length（因而讀序短）NextNext(3rd)-Gen

SequencingSingleMoleculeSequencing

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

(單分子合成

)UsingfluorescentlylabelednucleotidesReal-timesinglemolecule

fluorescencedetection

(單分子熒光實(shí)時(shí)監(jiān)測(cè))Immobilizedpolymerasevs.immobilizedDNA

template（聚合酶固定

vs.

DNA模板固定）SMS單分子測(cè)序流程Fragment

LibraryPreparation(DNA模板文庫(kù)制備）RandomPair-endImmobilization

ofFragment（DNA片段固定）Flat

surfaceParallelSequenceReaction（并行測(cè)序反應(yīng)）PolymeraseSimultaneousImageAcquisition（圖像采集與處理）FluorescenceSequenceReadandAssembly（序列拼接、組裝）ClonalAmplification（DNA片段擴(kuò)增）Emulsion

PCRBridgeamplificationCycleKeyChallengesofSingleMolecule

MonitoringSingle

Molecule

Detection

單分子監(jiān)測(cè)Detectionoffluorescencefromsinglemoleculein

real-time

(對(duì)單分子熒光進(jìn)行實(shí)時(shí)監(jiān)測(cè))Interference

of

background

fluorescence（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）epifluorescent

microscope（落射熒光顯微鏡）mercury

lampacooledCCD

cameraSingleMolecule

FluorescenceMicroscope（熒光顯微鏡）camera標(biāo)本接物鏡二色分光鏡TotalInternalReflectionFluorescence–

TIR全內(nèi)反射熒光顯微鏡技術(shù)Useevanescentwavetoilluminateandexcitefluorophoresinarestrictedregionadjacenttotheglass-water

interface

(用隱失波縮小熒光監(jiān)測(cè)范圍于玻水界面)Surfaceboundsinglemoleculescanbemonitored

with

high

signal

to

noise

(固相表面結(jié)合的單分子熒光信噪比提高)TIRF

Schematics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HeliScopeGenetic

AnalyzertSMS–trueSingleMolecule

SequencingA)incorporationoffluorescent

nucleotide（核苷酸合成）B)

washing（核苷酸沖洗）C)

imaging（熒光成像）D)

cleaving

label（去除熒光標(biāo)記）tSMS

Process單分子合成測(cè)序Acyclicprocessinvolvingmultiplerounds

of合成沖洗成像去除VisiGen(ABI)

BiotechnologiesSequencebysynthesiswithfluorescenceresonanceenergytransfer(SBS-FRET)

(熒光共振能量轉(zhuǎn)移+合成測(cè)序)Polymerase+donor

fluorophoreNucleotides+acceptor

fluorophoreEnergytransferduringnucleotide

incorporationAcceptoremitslightofaparticular

wavelengthColoroflightusestoidentifythe

baseForster(Fluorescent)ResonanceEnergyTransfer

(FRET)熒光共振能量轉(zhuǎn)移Energytransfermechanismbetweentwofluorescentdyes(donorandacceptor)throughlongrangedipoledipoleinteractions(T.

F?rster

1948)

(熒光供體、受體間的能量轉(zhuǎn)移）Onlyhappenwhenthedonorandacceptordyesareincloseproximity,<10nm,creatingaverysmallconfinementoftheacceptor

excitation

light

(近距能量轉(zhuǎn)移）OpticalDetection

System光學(xué)探測(cè)系統(tǒng)光譜分離全內(nèi)反射熒光激發(fā)光源3rd-Gen:Pacific

BiosciencesPacBioRS

IIWorkflow

工作流程度長(zhǎng)、精確度ZERO-MODE

WAVEGUIDEs零模波導(dǎo)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零模波導(dǎo)

PacificBiosciences’sSMRTchip

EachchipcontainsthousandsofZMWs.EachZMWisacylindricalhole

tensofnanometersindiameter,perforatingathinmetal(e.g.aluminum)filmsupportedbyatransparent

substrate激發(fā)光源WhentheZMWisilluminatedthroughthetransparentsubstratebylaserlight,thewavelengthofthelightistoolargeto

passthroughthewaveguide’saperture.Attenuatedevanescentlightfrom

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

fluorescence.隱失波?<<λ隱失波ZMWwithAnchored

Polymerase零模波導(dǎo)

+

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

occupanciesofapproximately30%wereobtainedforarangeofZMW

diameters.PolymeraseDNA固定聚合酶核苷酸熒光標(biāo)記Usefulinfocanbeobtainedfromsignal

pulses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PacBioRSIIAdvantagesLongreads:averagelengthsof4,200to

8,500Highaccuracy:with99%

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

damage.LeastGC

bias.Noamplification:noPCR

required.Advantageof

SMS單分子測(cè)序優(yōu)勢(shì)No

need

for

amplification（不用擴(kuò)增）High

information

density（信息密度高）Theoretical

limit

is

diffraction

limit

of

light，λ/2（光衍射極限）Error

rate

stay

flatvs.sequence

length（誤差率不隨鏈的延長(zhǎng)增加）Longer

readlength（讀序長(zhǎng)）Nodephasingissueduring

synthesisPotentialnewwayofdetectingmodified

nucleotide（提供修飾堿基檢測(cè)新方法）ChallengesforLight-based

SMS光學(xué)單分子測(cè)序面對(duì)的問(wèn)題Sequencing

Accuracy

（精確性）Single

molecule

fluorescence

detection

（單分子檢測(cè)）Fluorophore

blinking

（熒光間斷）Polymerase

fidelity

（聚合酶保真度）Sequencing

ReadLength

（讀長(zhǎng)）Photophysical

damage

（光損害）Photo-bleaching

to

fluorophore

（熒光團(tuán)漂泊）Damage

to

DNA

polymerase

（聚合酶損傷）Future

Generation（4th-Gen）Physical

method

andmeasurement（物理方法）Visualizingsequencingby

microspectroscopy（基于電子顯微鏡直接測(cè)序法）Nano-structurebased

measurement

(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）Still

in

infancy

（萌芽期）4th-Generation

Benefits四代優(yōu)勢(shì)Label

free（無(wú)標(biāo)記物）+amplification

free（無(wú)擴(kuò)增）No

need

for

optical

imaging

system（無(wú)需光學(xué)顯微鏡）Simple

data

processing

and

storage（數(shù)據(jù)處理簡(jiǎn)易）Lower

cost

(低成本)

+

high

speed（高速度）Microscopic

Sequencing（顯微鏡測(cè)序)SimplyVisualizing

BasesUsingscanningprobemicroscopes（SPM,

掃描探針顯微鏡）Atomic

force

microscope(AFM，原子力顯微鏡

)CoupledwithRamanspectrumScanning

tunneling

microscope

(STM，掃描隧道顯微鏡)Direct-readGeneticSequencingbyZSGeneticswithElectron

MicroscopeUse

polymerase

with

heavy

atom

labeled

（重原子標(biāo)記）nucleotidestosynthesisstrandtobedirectlyreadbyEMSolid-State

Nanopore（固體納米孔）IonBeam

SculpturedNanoporeinSiliconNitrideElectronBeamPunchedNanoporeonGrapheneBiological

Nanopore（生物納米孔）Nanopore

with

engineered

protein

(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）KeyChallengesforNanopore-Based

Sequencing納米孔測(cè)序的挑戰(zhàn)DetectionofindividualbasesasDNA

passing

through

(分辨不同堿基)ControloftraversingspeedofDNAchain

through

nanopore

(DNA鏈穿孔速度控制)NanoporeDetectionCurrent

Blockade

-

阻塞電流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(dá)NanoporeStrand

SequencingApplications應(yīng)用ComparisonofNGSPlatforms新一代測(cè)序平臺(tái)比較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新一代代測(cè)序平臺(tái)應(yīng)用對(duì)象Genomicanalysis

(DNA)GenomeorDNAsequencingGenomic

variant

discovery

（基因組變異的發(fā)掘）Genomicalterationand

modificationTranscriptomicanalysis(RNA)Transcript

profilingSmallRNA

analysisBiomarkerdiscoverybyProtona

(Protein)Proof-of-conceptworkinsequencingprotein-bound

oligonucleotide

aptamer

(蛋白/核酸適配體)EukaryoticGenomeSequencingProjects

（真核生物基因組）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人類基因組再測(cè)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

轉(zhuǎn)錄組WholeTranscriptomeShotgunSequencingorRNA-seqArevolutionarytoolfor

transcriptomicsPriortothis,microarrayisthetoolof

choiceUtilize

deep

sequencing cDNAwithNGSto

studytranscriptsmRNAtranscript

analysisDigital

gene

expression analysis(數(shù)碼基因差異表達(dá)）Alternative

mRNA

splice

forms

(非傳統(tǒng)RNA剪接）Discoveryandprofilingofgenome-widenon-coding

small

RNA

（非編碼RNA）ShortreadsareidealforstudyofnovelmiRNAandsiRNA(~30baselongwithregulatory

roles)Advantageof

RNA-seqEliminatetheneedforclonal

amplificationEmulsionPCRorbridge

amplificationNopriorknowledgeofgenome

sequencerequiredMicroarrayrequiresitforconstructionof

probesDigital

countingWiderdynamicrangeassequencinggets

deeperRelativelylowcost(comparingwitholdtechniques)Epigenomics(表觀基因組）Genome-wide

DNA-proteins

interaction（蛋白/核酸相互作用）Interactionoftranscriptionfactorwithitsdirecttargets

（轉(zhuǎn)錄因子）Genomic

profiling

of

histone

modification

（組蛋白修飾）Genomic

profiling

of

nucleosome

positions

（核小體定位）DNA

methylation

（DNA甲基化）ChIP-seqChromatinImmunoprecipatation-DirectSequencing染色質(zhì)免疫共沉淀-測(cè)序Proteinofinterestcross-linkedwith

DNATreatmentwith

formaldehydeDNA-proteincomplexsheartogiverisesmall

fr