測(cè)序技術(shù)基礎(chǔ)原理課件

測(cè)序技術(shù)基礎(chǔ)羅龍海2009-03-02測(cè)序技術(shù)基礎(chǔ)羅龍海Sanger測(cè)序技術(shù)原理第二代測(cè)序技術(shù)原理第三代測(cè)序技術(shù)原理Sanger測(cè)序技術(shù)原理1950196019701980199020002010測(cè)序技術(shù)發(fā)展史DevelopmentofSangerSequencing(1977)InventionofAutomatedFluorescentSequencer(1985)InventionofCapillarySequencer(1996)InventionofAppliedBiosystemsSolidSystem(2007)InventionofIlluminaGenomeAnalyzerSystem(2006)Inventionof454GS20Sequencer(2005)chemicaldegradationmethodbyMaxam-Gilbertmethod(1977)ChemicaldegradationmethodbyWhitfield(1954)InventionofHeliscopesinglemolecularsequencerInventionofSinglemoleculerealtime(SMRT)DNAsequencingInventionofNanoporesinglemolecularsequencing(OxfordNanoporecorporation)1950196019701980199020002010測(cè)序測(cè)序技術(shù)基礎(chǔ)原理課件測(cè)序技術(shù)基礎(chǔ)原理課件測(cè)序技術(shù)基礎(chǔ)原理課件Sanger測(cè)序法原理Dr.FredSanger"dideoxy"sequencingtechnique(Sangeretal.,1977)DNA雙脫氧鏈終止法測(cè)序FrederickSangerwasawardedtheprizeinboth1958and1980.

HeisthefourthpersonintheworldtohavebeenawardedtwoNobelPrizesandtheonlypersontoreceivebothinchemistry.

Sanger測(cè)序法原理Dr.FredSanger"d測(cè)序技術(shù)基礎(chǔ)原理課件測(cè)序技術(shù)基礎(chǔ)原理課件測(cè)序技術(shù)基礎(chǔ)原理課件IlluminaGenomeAnalyzerABISOLiDRoche454第二代測(cè)序技術(shù)IlluminaGenomeAnalyzer第二代測(cè)二代測(cè)序技術(shù)200520062007年份原理Pyrosequencing邊合成邊測(cè)序邊連接邊測(cè)序454SolexaSOLID二代測(cè)序技術(shù)200520062007年份原理PyrosequIlluminaSolexaABISOLiDRoche454第二代測(cè)序技術(shù)IlluminaSolexa第二代測(cè)序技術(shù)可逆阻斷技術(shù)可逆阻斷技術(shù)IlluminaSolexaFlowcellFlowcell一個(gè)flowcell

包括8個(gè)lanes

Lane1Lane8Eachlanecontainsmultipletiles–total100每個(gè)lane上有許多個(gè)tiles——共計(jì)100個(gè)（見上圖）Eachtileisimagedfourtimespercycle–oneimageperbase每個(gè)循環(huán)會(huì)對(duì)每個(gè)tile照4次相——每個(gè)堿基都會(huì)成像Imagefrom1tileIlluminaSolexaFlowcellFlowcGenomeAnalyzerAnalysisPipelineLibraryPreparationClusterStationGenomicmRNASmallRNAChIP-SeqIllumina/GAWorkflow

工作流程*GrowClusters*5hours*Orsplitprocessinstages*Safestoppingpoints*StartSequencing*ClusterDensityEvaluation*4imagespertilepercycle*Runtime:2-3days*Firecrest:ImageAnalysis*Bustard:Basecalling*Gerald:SequenceAlignment文庫(kù)構(gòu)建Cluster工作站基因組測(cè)序分析*生成“簇”*5小時(shí)*開始測(cè)序*序列簇的豐度檢測(cè)*一個(gè)循環(huán)每個(gè)tile照4張照片*2-3天*圖像分析*Bustard:basecalling*Gerald:序列分析?樣品預(yù)處理PCR成簇測(cè)序拼接分析5-6h6-8dGenomeAnalyzerAnalysisPipeli（純化的基因組DNA）（基因組DNA小片段小于800bp）（具有5’-磷酸末端的粘性片段）（去除未連接上的接頭）（修飾末端）（）（基因組DNA文庫(kù)）（純化連接產(chǎn)物）（）（）（）（基因組DNA小片段）（純化的基因組DNA）（基因組DNA小片段（具有5’-磷酸末27bp27bp27bp27bpGenomicDNAFragmentlibraryMate-pairedlibraryCreatelibraryofDNAfragmentsDNA片段的文庫(kù)構(gòu)建27bp27bp27bp27bpGenomicDNAFrClusterGeneration序列簇的產(chǎn)生PrepareDNAfragmentsLigateadaptersAttachsinglemoleculestosurfaceAmplifytoformclustersRandomarrayofclusters（Cluster的隨機(jī)排列）

100um~1000moleculesper~1umcluster

~20.000clusterspertile32-40millionclustersperexperiment

（1個(gè)cluster上有1000個(gè)分子

1個(gè)tile上有20,000個(gè)cluster

每個(gè)實(shí)驗(yàn)可完成3.2-4千萬個(gè)cluster)通過cluster將單分子放大、固定ClusterGeneration序列簇的產(chǎn)生PreparOHOHGraftedflowcelldiol

P7

P5

ClusterGeneration:AmplificationdioldiolTemplateHybridization（模板雜交）dioldiolInitialextensiondioldiol1stcycledenaturation（No.1循環(huán)：變性）1stcycleannealing（No.1循環(huán)：退火）dioldiol1stcycleextension（No.1循環(huán)：延伸）dioldioldioldiol2ndcycledenaturation2ndcycleannealingdioldioldiolOHOHGraftedflowcelldiolP7ClusterGeneration:Amplificationdioldioldiol2ndcycleextensionClusterAmplificationOHdioldiolOHPeriodateLinearization(高碘酸鹽，線性化)OHBlockingwithddNTP()(ddNTP阻斷末端)DenatureandHybridizationSBS3（變性、雜交）SBS——邊合成邊測(cè)序OHClusterGeneration:AmplificatSequencingBySynthesis邊合成邊測(cè)序1.Incorporation結(jié)合2.Scan掃描拍照3.Cleavage清洗SBSCycleSequencingBySynthesis1.IncoCAGTCATCACCTAGCGTA5’GTCAGTCAGTCAGT3’5’ Firstbaseincorporated第一個(gè)堿基合成上Cycle1: Addsequencingreagents加入合成所需反應(yīng)物 DetectSignal檢測(cè)熒光信號(hào) CleaveTerminatorandDye去掉末端封閉，染色Cycle2-n:AddsequencingreagentsandrepeatSequencingBySynthesis(SBS)?CAGTCATCACCTAGCGTA5’GTCAGTCAGTBaseCalling堿基識(shí)別123789456TTTTTTT

G

T…T

G

C

T

A

C

G

A

T…TheidentityofeachbaseofaclusterisreadofffromsequentialimagesBaseCalling123789456TTTTTPairedEndPairedEndPairedEnd雙末端測(cè)序、正反雙向測(cè)序SamplePreparation樣品前準(zhǔn)備ClusterGeneration分子簇的生成SequenceBySynthesis邊合成邊測(cè)序（用于組裝較大的Gene，一次最多讀100bp）PairedEndSamplePreparationSamplePreparation5’T3’A5’AT3’3’A5’T3’TA5’加雙末端SamplePreparation5’T3’A5’AT3’OHOHdiol

P7

P5GraftedFlowCellsSingleReadPeriodateLinearizationPairedEndUracilSpecificExcisionReagent(USER)

尿嘧啶特異性識(shí)別位點(diǎn)-P5formamidopyrimidineglycosylase(fpg)…糖基化酶-P38oxoG-P7

U-P5

OHOHU8oxo-G?OHOHdiolP7P5GrafteTemplatehybridizationUUOHOHGraftedflowcellU

P7

P5ClusterGeneration:InitialExtensionInitialextensionUU1stcycleDenaturationUU1stcycleannealingUU1stcycleextensionUU2ndcycledenaturationUU2ndcycleannealingUUUTemplateUUOHOHGraftedflowcen=25totalClusterGeneration:Amplification成簇2ndcycleextensionUUUClusterAmplificationUUP5Linearization(USER)胞嘧啶識(shí)別位點(diǎn)處切割BlockwithddNTPs末端ddNTPs封閉DenaturationandHybridizationSBS325個(gè)循環(huán)n=25ClusterGeneration:AmplifSequencing測(cè)序DenaturationandHybridizationSBS3SequencingFirstRead（NO.1次讀序）DenaturationandDe-Phosphorylation(PNK)變性和去磷酸作用OHOHResynthesisofP5StrandOHP7Linearization(fpg)OHBlockwithddNTPsDenaturationandHybridizationSBS8SequencingSecondRead（NO.2次讀序）Sequencing測(cè)序DenaturationandSequencingbiochemistryBase/RunTime/runReadlengthDominanterrortypeDNAcluster、Reversibleterminators、SequencingbySynthesis40-50Gb10days2*75bpSubstitutionIlluminaSolexa形成DNA簇；可逆阻斷技術(shù)邊合成邊測(cè)序（SBS）40-50G/10天/一個(gè)RUN讀長(zhǎng)：75bp（可雙向）錯(cuò)誤類型：替換SequencingbiochemistryBase/RIlluminasolexaABISOLiDRoche454IlluminasolexaSOLID測(cè)序技術(shù)SOLID測(cè)序技術(shù)AB/SOLIDWorkflow工作流程文庫(kù)制備EmulsionPCRBeadsEnrichment微珠沉積連接測(cè)序數(shù)據(jù)分析AB/SOLIDWorkflow工作流程文庫(kù)制備文庫(kù)制備EmulsionPCRBeadsEnrichment微珠沉積連接測(cè)序數(shù)據(jù)分析WorkFlow：文庫(kù)制備WorkFlow：序列可以用超聲波、機(jī)械剪切或酶解等方法，隨機(jī)或者定向的打斷成小片段序列可以用超聲波、機(jī)械剪切或酶解等方法，隨機(jī)或者定向的打斷成（大片段兩頭測(cè)序）“Mate-paired”步驟：環(huán)化——切割——加接頭——測(cè)序（大片段兩頭測(cè)序）“Mate-paired”步驟：環(huán)化——酶切為粘性末端對(duì)于復(fù)雜的分子環(huán)化，采用低濃度的模板濃度進(jìn)行連接酶切為粘性末端對(duì)于復(fù)雜的分子環(huán)化，采用測(cè)序技術(shù)基礎(chǔ)原理課件文庫(kù)制備EmulsionPCR微珠富集微珠沉積連接測(cè)序數(shù)據(jù)分析WorkFlow：文庫(kù)制備WorkFlow：2.EmulsionPCR+TemplatesEnzyme+dNTPsP1-coupledDNAbeads~100,000P1sitesperbeadStartwith2BillionbeadsperemulsionPolymerase100,000P1位點(diǎn)/每個(gè)bead2Billionbeads/每個(gè)emulsion2.EmulsionPCR+TemplatesEnzMixPCRaqueousphaseintoawater-in-oilemulsionandcarryoutemulsionPCRReactorwithtemplate,beadandPCRreagentsMineraloil+surfactantsMixPCRaqueousphaseintoawBeadscollectedfollowingemulsionPCR:Beadswithamplifiedproduct(~40KPCRproductsperbead)BeadswithnoproductP1P2P2Beadscollectedfollowingemul文庫(kù)制備EmulsionPCR微珠富集微珠沉積連接測(cè)序數(shù)據(jù)分析WorkFlow：文庫(kù)制備WorkFlow：3.Enrichment/微珠富集CentrifugeusingaGlycerolGradient甘油梯度離心Capturedbeads(+templates)insupernatantUncapturedbeads(notemplate)inpellet3.Enrichment/微珠富集Centrifugeu文庫(kù)制備EmulsionPCR微珠富集微珠沉積連接測(cè)序數(shù)據(jù)分析WorkFlow：文庫(kù)制備WorkFlow：4.Depositebeads3’-endmodificationBeadsattachedtoglasssurfaceinarandomarrayTemplatebeaddeposition4.Depositebeads3’-endmodi文庫(kù)制備EmulsionPCR微珠富集微珠沉積連接測(cè)序數(shù)據(jù)分析WorkFlow：文庫(kù)制備WorkFlow：ligase3’p5’universalseqprimerTemplateSequence5’3’AdapterOligoSequence

1μmbeadA-probe5’3’C-probe5’3’G-probe5’3’T-probe5’nnnnAzzznnnnCzzznnnnTzzznnnnGzzz1μmbeaduniversalseqprimerp5’5.SOLiD4-colorligationreactionligase3’5.SOLiD4-colorligationreactionTemplateSequence5’3’AdapterOligoSequence1μmbead1μmbeadligaseligase3’p5’universalseqprimeruniversalseqprimer5’3’C-probe5’3’G-probe5’3’T-probe5’A-probennnnAzzznnnnCzzznnnnTzzznnnnGzzzAp5’5.SOLiD4-colorligationreacA5TemplateSequence5’3’AdapterOligoSequence1μmbeaduniversalseqprimerA1μmbead6.SOLiD4-colorligationvisualizationATemplateSequence5’3’AdapteTemplateSequence5’3’AdapterOligoSequence1μmbead1μmbeadC20T15G25A5T107.SOLiD4-colorligationResetTemplateSequence5’3’Adapterligase8.SOLiD4-colorligation(1stcycleafterreset)ligase3’p5’universalseqprimern-1TemplateSequence5’3’AdapterOligoSequence1μmbeaduniversalseqprimern-1TA-probe5’3’C-probe5’3’G-probe5’3’T-probe5’nnnnAzzznnnnCzzznnnnTzzznnnnGzzz1μmbeadp5’ligase8.SOLiD4-colorligatio測(cè)序技術(shù)基礎(chǔ)原理課件Consequencesof2BasePairEncoding

Detectingasinglecolordoesnotindicateabase

EachreadingcontainsinformationfromtwobasesTodecodethebasesyoumustknowoneofthebasesinthesequenceACGTACGT2ndBase1stBaseConsequencesof2BasePairEnACGTACGT2ndBase1stBaseIfknowfirstbaseisanAthenimmediatelyitdecodes2ndbase.ThismustbeanAasBluetranslates2ndbaseAiffirstbaseAAACCGGTTACCAGTTGACCAGTTGAACCGGTTAACCGGTTAGCTGATCAGCTGATCAGCTGATCATCGGCTAExample:ACGTACGT2ndBase1stBaseIfknoABISOLiDSequencingbiochemistryBase/RunTime/runReadlengthDominanterrortypeEmulsionPCRSequencingbyligation50Gb>10days2*50bpSubstitutionEmulsionPCR邊連接邊測(cè)序（SBL）50G/大于10天/一個(gè)RUN讀長(zhǎng)：50bp（可雙向）錯(cuò)誤類型：替換ABISOLiDSequencingbiochemisIlluminasolexaABISOLiDRoche454IlluminasolexaGenomeSequencer20Syste（2005）GenomeSequencerFLXSyste（2006）GSFLXTitanium（2008）發(fā)展歷程：GenomeSequencer20Syste（20061emPCRSequencingDNALibraryPreparationDNALibraryPreparationGenomefragmentedbynebulizationAdaptorligationsstDNAlibrarycreatedwithadaptersA/Bfragmentsselectedusingavidin-biotinpurificationEmulsionPCRAmplificationAnnealsstDNAtoanexcessofDNAcapturebeadsEmulsifybeadsandPCRreagentsinwater-in-oilmicroreactorsClonalamplificationoccursinsidemicroreactorsSequencingBySynthesisLoadbeadsintoPicoTiter?Plate

SequencingbysynthesisPhotonsGeneratedareCapturedbyCameraSequencingImageCreatedRoche/454GSFLXWorkflow61emPCRSequencingDNALibraryDNsstDNAlibrarygDNAGenomefragmentedbynebulizationNocloning;nocolonypickingsstDNAlibrarycreatedwithadaptersA/Bfragmentsselectedusingavidin-biotinpurification

1.DNAlibrarypreparationsstDNAlibrarygDNAGenomefragm2.EmulsionBasedClonalAmplificationClonally-amplifiedsstDNAattachedtobeadsstDNAlibraryAnnealsstDNAtoanexcessofDNAcapturebeadsEmulsifybeadsandPCRreagentsinwater-in-oilmicroreactorsBreakmicroreactors,enrichforDNA-positivebeadsClonalamplificationoccursinsidemicroreactors2.EmulsionBasedClonalAmplif3.LoadingDNABeadsintothePicoTiter?Plate3.LoadingDNABeadsintothePdNTPPPiPPi+APSATPATP+LuciferinluciferaseOxyluciferin+Light4.SequencingdNTPPPi4.SequenciThesequencinginstrumentconsistsofthefollowingmajorsubsystems:(a)afluidicassembly，(b)aflowchamberthatincludesthewell-containingfibre-opticslide，(c)aCCDcamera-basedimagingassembly，andacomputerthatprovidesthenecessaryuserinterfaceandinstrumentcontrol.ThesequencinginstrumentconsRoche454SequencingbiochemistryBase/RunTime/runReadlengthDominanterrortypeEmulsionPCRPolymerasepyrosequencing0.4-0.6Gb10hrs400bpInsertion&Deletion微乳液PCR聚合測(cè)序0.4-0.6G/10h

/一個(gè)RUN讀長(zhǎng)：400bpMax：800bp錯(cuò)誤類型：插入&缺失Roche454Sequencingbiochemis第二代測(cè)序技術(shù)小結(jié)454測(cè)序儀：

454測(cè)序儀使用的方法，經(jīng)微乳液PCR發(fā)擴(kuò)增后，攜帶有大量模板分子的微珠被放置到芯片上的微孔中。隨后使用焦磷酸法測(cè)序，每一輪測(cè)序反應(yīng)都會(huì)摻入一個(gè)核苷酸，隨后加入反應(yīng)試劑熒光素和腺苷酰硫酸。這樣在每一個(gè)小孔中每當(dāng)有聚合酶將核苷酸摻入到模板上都會(huì)發(fā)光。最后用腺苷三磷酸雙磷酸酶洗滌去掉多余的核苷酸。(對(duì)重復(fù)序列如polyA的測(cè)定不準(zhǔn)確，因熒光信號(hào)具有累加效果)Solexa測(cè)序儀：Solexa測(cè)序儀使用橋式PCR直接在芯片進(jìn)行模板擴(kuò)增，然后同時(shí)加入四種經(jīng)過修飾的脫氧核苷酸，每一個(gè)核苷酸都攜帶一種熒光集團(tuán)和一個(gè)可被去除的終止基團(tuán)。經(jīng)過修飾的DNA聚合酶催化引物延伸測(cè)序反應(yīng)。采集圖像、然后切除熒光標(biāo)記基團(tuán)和終止基團(tuán)，重復(fù)上述反應(yīng)，完成測(cè)序。（邊合成邊測(cè)序）Solid測(cè)序儀：Solid測(cè)序儀使用微乳液PCR法擴(kuò)增模板片段，然后吸附有大量擴(kuò)增片段的直徑1um的磁珠倍制成高密度測(cè)序芯片，借助使用連接酶而不是聚合酶測(cè)序法完成測(cè)序。在solid測(cè)序一中，每一次反應(yīng)都會(huì)在引物末端加上一個(gè)熒光標(biāo)記的8bp的探針，在探針中央的兩個(gè)堿基上標(biāo)記有熒光基團(tuán)，探針被連接上之后發(fā)出熒光，隨后熒光基團(tuán)部分被切除，重新系下一輪反應(yīng)。（邊連接邊測(cè)序）第二代測(cè)序技術(shù)小結(jié)454測(cè)序儀：454測(cè)序儀使用的方法，經(jīng)

第一代測(cè)序技術(shù)versus

第二代測(cè)序技術(shù)第一代測(cè)序技術(shù)CurrentpopularsequencingplatformCompanyFormatReadLength(bases)讀長(zhǎng)(bp)ExpectedThroughput

(Gb/Run)測(cè)序通量AppliedBiosystemsCapillaryelectrophoresis10003-4MSolexaParallelmicrochip75+7550SolidSequencingbyligation50+5050454LifeSciencesParallelbeadarray200(400)+200(400)0.4Currentpopularsequencingpla第三代測(cè)序技術(shù)第三代測(cè)序技術(shù)Heliscope單分子測(cè)序儀--HelicosBiosciences測(cè)序原理：邊合成邊測(cè)序特點(diǎn)：無需對(duì)待測(cè)模板進(jìn)行擴(kuò)增，采用高靈敏度的熒光探測(cè)儀，直接對(duì)單鏈的DNA模板進(jìn)行合成測(cè)序，序列讀長(zhǎng)為24到70個(gè)堿基，平均讀長(zhǎng)為32個(gè)堿基。流程：（1）待測(cè)文庫(kù)片段化；（2）3’端加polyA尾，并與固定在芯片上的polyT進(jìn)行雜交，將待測(cè)模板固定到芯片上，制成測(cè)序芯片。（3）通過DNA聚合酶將熒光標(biāo)記的單核苷酸滲入到引物上，每一輪反應(yīng)加入一種dNTP。（4）采集熒光信號(hào)，切除熒光標(biāo)記集團(tuán)，進(jìn)行下一輪測(cè)序反應(yīng)。Heliscope單分子測(cè)序儀測(cè)序原理：邊合成邊測(cè)序斯坦福大學(xué)的科學(xué)家最近利用HelicosBiosciences的Heliscope單分子測(cè)序儀，對(duì)一名白人男子的基因組進(jìn)行了測(cè)序，文章發(fā)表在最新一期的《NatureBiotechnology》在線版上。利用一臺(tái)Heliscope測(cè)序儀和4次數(shù)據(jù)收集運(yùn)行，完成了此次測(cè)序。研究人員報(bào)告稱，他們產(chǎn)生了數(shù)十億個(gè)Heliscope序列讀取，覆蓋了90%的人參考基因組，覆蓋度達(dá)28倍。序列讀長(zhǎng)為24到70個(gè)堿基，平均讀長(zhǎng)為32個(gè)堿基。到目前為止，他們已經(jīng)鑒定出280萬個(gè)SNP和752個(gè)拷貝數(shù)變異。測(cè)序花了4個(gè)星期的時(shí)間，試劑花費(fèi)為48000美元

斯坦福大學(xué)的科學(xué)家最近利用HelicosBiosciencSinglemoleculerealtime(SMRT)DNA測(cè)序技術(shù)—PacificBiosciences（1）以SMRT芯片載體：帶有3000個(gè)直徑為70nm左右的納米級(jí)小孔的金屬片，將DNA聚合酶、待測(cè)序列和不同熒光標(biāo)記的dNTP放入到ZMW孔中，進(jìn)行合成反應(yīng)。（2）SMRT技術(shù)的測(cè)序速度很快，可達(dá)到每秒大約10個(gè)dNTP。、它實(shí)現(xiàn)了DNA聚合酶內(nèi)在自身的反應(yīng)速度，一秒可以測(cè)10個(gè)堿基，測(cè)序速度是化學(xué)法測(cè)序的2萬倍。（3）它實(shí)現(xiàn)了DNA聚合酶內(nèi)在自身的processivity（延續(xù)性，也就是DNA聚合酶一次可以合成很長(zhǎng)的片段），一個(gè)反應(yīng)就可以測(cè)非常長(zhǎng)的序列。二代測(cè)序現(xiàn)在可以測(cè)到上百個(gè)堿基，但是三代測(cè)序現(xiàn)在就可以測(cè)幾千個(gè)堿基。這為基因組的重復(fù)序列的拼接提供了非常好的條件。（4）它的精度非常高，達(dá)到99.9999%。Singlemoleculerealtime(SMRT納米孔單分子技術(shù)--OxfordNanopore公司測(cè)序原理：不同堿基產(chǎn)生的電信號(hào)進(jìn)行測(cè)序。步驟：特殊材料制成的納米孔，孔內(nèi)共價(jià)結(jié)合有分子接頭環(huán)糊精，核酸外切酶切割單鏈DNA時(shí)，被切割下來的堿基落入納米孔，并與環(huán)糊精相互作用，短暫影響流過納米孔的電流強(qiáng)度，電流強(qiáng)度的變化幅度成為每種堿基的檢測(cè)特征。堿基在納米孔中的平均停留時(shí)間是毫秒級(jí)的，一定強(qiáng)度的電壓可保證在電信號(hào)記錄后將堿基從納米孔中清除。獨(dú)特特點(diǎn)：直接讀取甲基化的胞嘧啶。納米孔單分子技術(shù)測(cè)序原理：不同堿基產(chǎn)生的電信號(hào)進(jìn)行測(cè)序。測(cè)序技術(shù)基礎(chǔ)原理課件測(cè)序技術(shù)基礎(chǔ)羅龍海2009-03-02測(cè)序技術(shù)基礎(chǔ)羅龍海Sanger測(cè)序技術(shù)原理第二代測(cè)序技術(shù)原理第三代測(cè)序技術(shù)原理Sanger測(cè)序技術(shù)原理1950196019701980199020002010測(cè)序技術(shù)發(fā)展史DevelopmentofSangerSequencing(1977)InventionofAutomatedFluorescentSequencer(1985)InventionofCapillarySequencer(1996)InventionofAppliedBiosystemsSolidSystem(2007)InventionofIlluminaGenomeAnalyzerSystem(2006)Inventionof454GS20Sequencer(2005)chemicaldegradationmethodbyMaxam-Gilbertmethod(1977)ChemicaldegradationmethodbyWhitfield(1954)InventionofHeliscopesinglemolecularsequencerInventionofSinglemoleculerealtime(SMRT)DNAsequencingInventionofNanoporesinglemolecularsequencing(OxfordNanoporecorporation)1950196019701980199020002010測(cè)序測(cè)序技術(shù)基礎(chǔ)原理課件測(cè)序技術(shù)基礎(chǔ)原理課件測(cè)序技術(shù)基礎(chǔ)原理課件Sanger測(cè)序法原理Dr.FredSanger"dideoxy"sequencingtechnique(Sangeretal.,1977)DNA雙脫氧鏈終止法測(cè)序FrederickSangerwasawardedtheprizeinboth1958and1980.

HeisthefourthpersonintheworldtohavebeenawardedtwoNobelPrizesandtheonlypersontoreceivebothinchemistry.

Sanger測(cè)序法原理Dr.FredSanger"d測(cè)序技術(shù)基礎(chǔ)原理課件測(cè)序技術(shù)基礎(chǔ)原理課件測(cè)序技術(shù)基礎(chǔ)原理課件IlluminaGenomeAnalyzerABISOLiDRoche454第二代測(cè)序技術(shù)IlluminaGenomeAnalyzer第二代測(cè)二代測(cè)序技術(shù)200520062007年份原理Pyrosequencing邊合成邊測(cè)序邊連接邊測(cè)序454SolexaSOLID二代測(cè)序技術(shù)200520062007年份原理PyrosequIlluminaSolexaABISOLiDRoche454第二代測(cè)序技術(shù)IlluminaSolexa第二代測(cè)序技術(shù)可逆阻斷技術(shù)可逆阻斷技術(shù)IlluminaSolexaFlowcellFlowcell一個(gè)flowcell

包括8個(gè)lanes

Lane1Lane8Eachlanecontainsmultipletiles–total100每個(gè)lane上有許多個(gè)tiles——共計(jì)100個(gè)（見上圖）Eachtileisimagedfourtimespercycle–oneimageperbase每個(gè)循環(huán)會(huì)對(duì)每個(gè)tile照4次相——每個(gè)堿基都會(huì)成像Imagefrom1tileIlluminaSolexaFlowcellFlowcGenomeAnalyzerAnalysisPipelineLibraryPreparationClusterStationGenomicmRNASmallRNAChIP-SeqIllumina/GAWorkflow

工作流程*GrowClusters*5hours*Orsplitprocessinstages*Safestoppingpoints*StartSequencing*ClusterDensityEvaluation*4imagespertilepercycle*Runtime:2-3days*Firecrest:ImageAnalysis*Bustard:Basecalling*Gerald:SequenceAlignment文庫(kù)構(gòu)建Cluster工作站基因組測(cè)序分析*生成“簇”*5小時(shí)*開始測(cè)序*序列簇的豐度檢測(cè)*一個(gè)循環(huán)每個(gè)tile照4張照片*2-3天*圖像分析*Bustard:basecalling*Gerald:序列分析?樣品預(yù)處理PCR成簇測(cè)序拼接分析5-6h6-8dGenomeAnalyzerAnalysisPipeli（純化的基因組DNA）（基因組DNA小片段小于800bp）（具有5’-磷酸末端的粘性片段）（去除未連接上的接頭）（修飾末端）（）（基因組DNA文庫(kù)）（純化連接產(chǎn)物）（）（）（）（基因組DNA小片段）（純化的基因組DNA）（基因組DNA小片段（具有5’-磷酸末27bp27bp27bp27bpGenomicDNAFragmentlibraryMate-pairedlibraryCreatelibraryofDNAfragmentsDNA片段的文庫(kù)構(gòu)建27bp27bp27bp27bpGenomicDNAFrClusterGeneration序列簇的產(chǎn)生PrepareDNAfragmentsLigateadaptersAttachsinglemoleculestosurfaceAmplifytoformclustersRandomarrayofclusters（Cluster的隨機(jī)排列）

100um~1000moleculesper~1umcluster

~20.000clusterspertile32-40millionclustersperexperiment

（1個(gè)cluster上有1000個(gè)分子

1個(gè)tile上有20,000個(gè)cluster

每個(gè)實(shí)驗(yàn)可完成3.2-4千萬個(gè)cluster)通過cluster將單分子放大、固定ClusterGeneration序列簇的產(chǎn)生PreparOHOHGraftedflowcelldiol

P7

P5

ClusterGeneration:AmplificationdioldiolTemplateHybridization（模板雜交）dioldiolInitialextensiondioldiol1stcycledenaturation（No.1循環(huán)：變性）1stcycleannealing（No.1循環(huán)：退火）dioldiol1stcycleextension（No.1循環(huán)：延伸）dioldioldioldiol2ndcycledenaturation2ndcycleannealingdioldioldiolOHOHGraftedflowcelldiolP7ClusterGeneration:Amplificationdioldioldiol2ndcycleextensionClusterAmplificationOHdioldiolOHPeriodateLinearization(高碘酸鹽，線性化)OHBlockingwithddNTP()(ddNTP阻斷末端)DenatureandHybridizationSBS3（變性、雜交）SBS——邊合成邊測(cè)序OHClusterGeneration:AmplificatSequencingBySynthesis邊合成邊測(cè)序1.Incorporation結(jié)合2.Scan掃描拍照3.Cleavage清洗SBSCycleSequencingBySynthesis1.IncoCAGTCATCACCTAGCGTA5’GTCAGTCAGTCAGT3’5’ Firstbaseincorporated第一個(gè)堿基合成上Cycle1: Addsequencingreagents加入合成所需反應(yīng)物 DetectSignal檢測(cè)熒光信號(hào) CleaveTerminatorandDye去掉末端封閉，染色Cycle2-n:AddsequencingreagentsandrepeatSequencingBySynthesis(SBS)?CAGTCATCACCTAGCGTA5’GTCAGTCAGTBaseCalling堿基識(shí)別123789456TTTTTTT

G

T…T

G

C

T

A

C

G

A

T…TheidentityofeachbaseofaclusterisreadofffromsequentialimagesBaseCalling123789456TTTTTPairedEndPairedEndPairedEnd雙末端測(cè)序、正反雙向測(cè)序SamplePreparation樣品前準(zhǔn)備ClusterGeneration分子簇的生成SequenceBySynthesis邊合成邊測(cè)序（用于組裝較大的Gene，一次最多讀100bp）PairedEndSamplePreparationSamplePreparation5’T3’A5’AT3’3’A5’T3’TA5’加雙末端SamplePreparation5’T3’A5’AT3’OHOHdiol

P7

P5GraftedFlowCellsSingleReadPeriodateLinearizationPairedEndUracilSpecificExcisionReagent(USER)

尿嘧啶特異性識(shí)別位點(diǎn)-P5formamidopyrimidineglycosylase(fpg)…糖基化酶-P38oxoG-P7

U-P5

OHOHU8oxo-G?OHOHdiolP7P5GrafteTemplatehybridizationUUOHOHGraftedflowcellU

P7

P5ClusterGeneration:InitialExtensionInitialextensionUU1stcycleDenaturationUU1stcycleannealingUU1stcycleextensionUU2ndcycledenaturationUU2ndcycleannealingUUUTemplateUUOHOHGraftedflowcen=25totalClusterGeneration:Amplification成簇2ndcycleextensionUUUClusterAmplificationUUP5Linearization(USER)胞嘧啶識(shí)別位點(diǎn)處切割BlockwithddNTPs末端ddNTPs封閉DenaturationandHybridizationSBS325個(gè)循環(huán)n=25ClusterGeneration:AmplifSequencing測(cè)序DenaturationandHybridizationSBS3SequencingFirstRead（NO.1次讀序）DenaturationandDe-Phosphorylation(PNK)變性和去磷酸作用OHOHResynthesisofP5StrandOHP7Linearization(fpg)OHBlockwithddNTPsDenaturationandHybridizationSBS8SequencingSecondRead（NO.2次讀序）Sequencing測(cè)序DenaturationandSequencingbiochemistryBase/RunTime/runReadlengthDominanterrortypeDNAcluster、Reversibleterminators、SequencingbySynthesis40-50Gb10days2*75bpSubstitutionIlluminaSolexa形成DNA簇；可逆阻斷技術(shù)邊合成邊測(cè)序（SBS）40-50G/10天/一個(gè)RUN讀長(zhǎng)：75bp（可雙向）錯(cuò)誤類型：替換SequencingbiochemistryBase/RIlluminasolexaABISOLiDRoche454IlluminasolexaSOLID測(cè)序技術(shù)SOLID測(cè)序技術(shù)AB/SOLIDWorkflow工作流程文庫(kù)制備EmulsionPCRBeadsEnrichment微珠沉積連接測(cè)序數(shù)據(jù)分析AB/SOLIDWorkflow工作流程文庫(kù)制備文庫(kù)制備EmulsionPCRBeadsEnrichment微珠沉積連接測(cè)序數(shù)據(jù)分析WorkFlow：文庫(kù)制備WorkFlow：序列可以用超聲波、機(jī)械剪切或酶解等方法，隨機(jī)或者定向的打斷成小片段序列可以用超聲波、機(jī)械剪切或酶解等方法，隨機(jī)或者定向的打斷成（大片段兩頭測(cè)序）“Mate-paired”步驟：環(huán)化——切割——加接頭——測(cè)序（大片段兩頭測(cè)序）“Mate-paired”步驟：環(huán)化——酶切為粘性末端對(duì)于復(fù)雜的分子環(huán)化，采用低濃度的模板濃度進(jìn)行連接酶切為粘性末端對(duì)于復(fù)雜的分子環(huán)化，采用測(cè)序技術(shù)基礎(chǔ)原理課件文庫(kù)制備EmulsionPCR微珠富集微珠沉積連接測(cè)序數(shù)據(jù)分析WorkFlow：文庫(kù)制備WorkFlow：2.EmulsionPCR+TemplatesEnzyme+dNTPsP1-coupledDNAbeads~100,000P1sitesperbeadStartwith2BillionbeadsperemulsionPolymerase100,000P1位點(diǎn)/每個(gè)bead2Billionbeads/每個(gè)emulsion2.EmulsionPCR+TemplatesEnzMixPCRaqueousphaseintoawater-in-oilemulsionandcarryoutemulsionPCRReactorwithtemplate,beadandPCRreagentsMineraloil+surfactantsMixPCRaqueousphaseintoawBeadscollectedfollowingemulsionPCR:Beadswithamplifiedproduct(~40KPCRproductsperbead)BeadswithnoproductP1P2P2Beadscollectedfollowingemul文庫(kù)制備EmulsionPCR微珠富集微珠沉積連接測(cè)序數(shù)據(jù)分析WorkFlow：文庫(kù)制備WorkFlow：3.Enrichment/微珠富集CentrifugeusingaGlycerolGradient甘油梯度離心Capturedbeads(+templates)insupernatantUncapturedbeads(notemplate)inpellet3.Enrichment/微珠富集Centrifugeu文庫(kù)制備EmulsionPCR微珠富集微珠沉積連接測(cè)序數(shù)據(jù)分析WorkFlow：文庫(kù)制備WorkFlow：4.Depositebeads3’-endmodificationBeadsattachedtoglasssurfaceinarandomarrayTemplatebeaddeposition4.Depositebeads3’-endmodi文庫(kù)制備EmulsionPCR微珠富集微珠沉積連接測(cè)序數(shù)據(jù)分析WorkFlow：文庫(kù)制備WorkFlow：ligase3’p5’universalseqprimerTemplateSequence5’3’AdapterOligoSequence

1μmbeadA-probe5’3’C-probe5’3’G-probe5’3’T-probe5’nnnnAzzznnnnCzzznnnnTzzznnnnGzzz1μmbeaduniversalseqprimerp5’5.SOLiD4-colorligationreactionligase3’5.SOLiD4-colorligationreactionTemplateSequence5’3’AdapterOligoSequence1μmbead1μmbeadligaseligase3’p5’universalseqprimeruniversalseqprimer5’3’C-probe5’3’G-probe5’3’T-probe5’A-probennnnAzzznnnnCzzznnnnTzzznnnnGzzzAp5’5.SOLiD4-colorligationreacA5TemplateSequence5’3’AdapterOligoSequence1μmbeaduniversalseqprimerA1μmbead6.SOLiD4-colorligationvisualizationATemplateSequence5’3’AdapteTemplateSequence5’3’AdapterOligoSequence1μmbead1μmbeadC20T15G25A5T107.SOLiD4-colorligationResetTemplateSequence5’3’Adapterligase8.SOLiD4-colorligation(1stcycleafterreset)ligase3’p5’universalseqprimern-1TemplateSequence5’3’AdapterOligoSequence1μmbeaduniversalseqprimern-1TA-probe5’3’C-probe5’3’G-probe5’3’T-probe5’nnnnAzzznnnnCzzznnnnTzzznnnnGzzz1μmbeadp5’ligase8.SOLiD4-colorligatio測(cè)序技術(shù)基礎(chǔ)原理課件Consequencesof2BasePairEncoding

Detectingasinglecolordoesnotindicateabase

EachreadingcontainsinformationfromtwobasesTodecodethebasesyoumustknowoneofthebasesinthesequenceACGTACGT2ndBase1stBaseConsequencesof2BasePairEnACGTACGT2ndBase1stBaseIfknowfirstbaseisanAthenimmediatelyitdecodes2ndbase.ThismustbeanAasBluetranslates2ndbaseAiffirstbaseAAACCGGTTACCAGTTGACCAGTTGAACCGGTTAACCGGTTAGCTGATCAGCTGATCAGCTGATCATCGGCTAExample:ACGTACGT2ndBase1stBaseIfknoABISOLiDSequencingbiochemistryBase/RunTime/runReadlengthDominanterrortypeEmulsionPCRSequencingbyligation50Gb>10days2*50bpSubstitutionEmulsionPCR邊連接邊測(cè)序（SBL）50G/大于10天/一個(gè)RUN讀長(zhǎng)：50bp（可雙向）錯(cuò)誤類型：替換ABISOLiDSequencingbiochemisIlluminasolexaABISOLiDRoche454IlluminasolexaGenomeSequencer20Syste（2005）GenomeSequencerFLXSyste（2006）GSFLXTitanium（2008）發(fā)展歷程：GenomeSequencer20Syste（200137emPCRSequencingDNALibraryPreparationDNALibraryPreparationGenomefragmentedbynebulizationAdaptorligationsstDNAlibrarycreatedwithadaptersA/Bfragmentsselectedusingavidin-biotinpurificationEmulsionPCRAmplificationAnnealsstDNAtoanexcessofDNAcapturebeadsEmulsifybeadsandPCRreagentsinwater-in-oilmicroreactorsClonalamplificationoccursinsidemicroreactorsSequencingBySynthesisLoadbeadsintoPicoTiter?Plate

SequencingbysynthesisPhotonsGeneratedareCapturedbyCameraSequencingImageCreatedRoche/454GSFLXWorkflow61emPCRSequencingDNALibraryDNsstDNAlibrarygDNAGenomefragmentedbynebulizationNocloning;nocolonypickingsstDNAlibrarycreatedwithadaptersA/Bfragmentsselectedusingavidin-biotinpurification

1.DNAlibrarypreparationsstDNAlibrarygDNAGenomefragm2.EmulsionBasedClonalAmplificationClonally-amplifiedsstDNAattachedtobeadsstDNAlibraryAnnealsstDNAtoanexcessofDNAcapturebeadsEmulsifybeadsandPCRreagentsinwater-in-oilmicroreactorsBreakmicroreactors,enrichforDNA-positivebeadsClonalamplificationoccursinsidemicroreactors2.EmulsionBasedClonalAmplif3.LoadingDNABeadsintothePicoTiter?Plate3.LoadingDNABeadsintothePdNTPPPiPPi+APSATPATP+LuciferinluciferaseOxyluciferin+Light4.SequencingdNTPPPi4.SequenciThesequencinginst