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

HeisthefourthpersonintheworldtohavebeenawardedtwoNobelPrizesandtheonlypersontoreceivebothinchemistry.

Sanger測序法原理Dr.FredSanger"d測序技術基礎原理課件測序技術基礎原理課件測序技術基礎原理課件IlluminaGenomeAnalyzerABISOLiDRoche454第二代測序技術IlluminaGenomeAnalyzer第二代測二代測序技術200520062007年份原理Pyrosequencing邊合成邊測序邊連接邊測序454SolexaSOLID二代測序技術200520062007年份原理PyrosequIlluminaSolexaABISOLiDRoche454第二代測序技術IlluminaSolexa第二代測序技術可逆阻斷技術可逆阻斷技術IlluminaSolexaFlowcellFlowcell一個flowcell

包括8個lanes

Lane1Lane8Eachlanecontainsmultipletiles–total100每個lane上有許多個tiles——共計100個（見上圖）Eachtileisimagedfourtimespercycle–oneimageperbase每個循環會對每個tile照4次相——每個堿基都會成像Imagefrom1tileIlluminaSolexaFlowcellFlowcGenomeAnalyzerAnalysisPipelineLibraryPreparationClusterStationGenomicmRNASmallRNAChIP-SeqIllumina/GAWorkflow

工作流程*GrowClusters*5hours*Orsplitprocessinstages*Safestoppingpoints*StartSequencing*ClusterDensityEvaluation*4imagespertilepercycle*Runtime:2-3days*Firecrest:ImageAnalysis*Bustard:Basecalling*Gerald:SequenceAlignment文庫構建Cluster工作站基因組測序分析*生成“簇”*5小時*開始測序*序列簇的豐度檢測*一個循環每個tile照4張照片*2-3天*圖像分析*Bustard:basecalling*Gerald:序列分析?樣品預處理PCR成簇測序拼接分析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）（純化連接產物）（）（）（）（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片段的文庫構建27bp27bp27bp27bpGenomicDNAFrClusterGeneration序列簇的產生PrepareDNAfragmentsLigateadaptersAttachsinglemoleculestosurfaceAmplifytoformclustersRandomarrayofclusters（Cluster的隨機排列）

100um~1000moleculesper~1umcluster

~20.000clusterspertile32-40millionclustersperexperiment

（1個cluster上有1000個分子

1個tile上有20,000個cluster

每個實驗可完成3.2-4千萬個cluster)通過cluster將單分子放大、固定ClusterGeneration序列簇的產生PreparOHOHGraftedflowcelldiol

P7

P5

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

G

T…T

G

C

T

A

C

G

A

T…TheidentityofeachbaseofaclusterisreadofffromsequentialimagesBaseCalling123789456TTTTTPairedEndPairedEndPairedEnd雙末端測序、正反雙向測序SamplePreparation樣品前準備ClusterGeneration分子簇的生成SequenceBySynthesis邊合成邊測序（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)

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

U-P5

OHOHU8oxo-G?OHOHdiolP7P5GrafteTemplatehybridizationUUOHOHGraftedflowcellU

P7

P5ClusterGeneration:InitialExtensionInitialextensionUU1stcycleDenaturationUU1stcycleannealingUU1stcycleextensionUU2ndcycledenaturationUU2ndcycleannealingUUUTemplateUUOHOHGraftedflowcen=25totalClusterGeneration:Amplification成簇2ndcycleextensionUUUClusterAmplificationUUP5Linearization(USER)胞嘧啶識別位點處切割BlockwithddNTPs末端ddNTPs封閉DenaturationandHybridizationSBS325個循環n=25ClusterGeneration:AmplifSequencing測序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測序DenaturationandSequencingbiochemistryBase/RunTime/runReadlengthDominanterrortypeDNAcluster、Reversibleterminators、SequencingbySynthesis40-50Gb10days2*75bpSubstitutionIlluminaSolexa形成DNA簇；可逆阻斷技術邊合成邊測序（SBS）40-50G/10天/一個RUN讀長：75bp（可雙向）錯誤類型：替換SequencingbiochemistryBase/RIlluminasolexaABISOLiDRoche454IlluminasolexaSOLID測序技術SOLID測序技術AB/SOLIDWorkflow工作流程文庫制備EmulsionPCRBeadsEnrichment微珠沉積連接測序數據分析AB/SOLIDWorkflow工作流程文庫制備文庫制備EmulsionPCRBeadsEnrichment微珠沉積連接測序數據分析WorkFlow：文庫制備WorkFlow：序列可以用超聲波、機械剪切或酶解等方法，隨機或者定向的打斷成小片段序列可以用超聲波、機械剪切或酶解等方法，隨機或者定向的打斷成（大片段兩頭測序）“Mate-paired”步驟：環化——切割——加接頭——測序（大片段兩頭測序）“Mate-paired”步驟：環化——酶切為粘性末端對于復雜的分子環化，采用低濃度的模板濃度進行連接酶切為粘性末端對于復雜的分子環化，采用測序技術基礎原理課件文庫制備EmulsionPCR微珠富集微珠沉積連接測序數據分析WorkFlow：文庫制備WorkFlow：2.EmulsionPCR+TemplatesEnzyme+dNTPsP1-coupledDNAbeads~100,000P1sitesperbeadStartwith2BillionbeadsperemulsionPolymerase100,000P1位點/每個bead2Billionbeads/每個emulsion2.EmulsionPCR+TemplatesEnzMixPCRaqueousphaseintoawater-in-oilemulsionandcarryoutemulsionPCRReactorwithtemplate,beadandPCRreagentsMineraloil+surfactantsMixPCRaqueousphaseintoawBeadscollectedfollowingemulsionPCR:Beadswithamplifiedproduct(~40KPCRproductsperbead)BeadswithnoproductP1P2P2Beadscollectedfollowingemul文庫制備EmulsionPCR微珠富集微珠沉積連接測序數據分析WorkFlow：文庫制備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文庫制備EmulsionPCR微珠富集微珠沉積連接測序數據分析WorkFlow：文庫制備WorkFlow：4.Depositebeads3’-endmodificationBeadsattachedtoglasssurfaceinarandomarrayTemplatebeaddeposition4.Depositebeads3’-endmodi文庫制備EmulsionPCR微珠富集微珠沉積連接測序數據分析WorkFlow：文庫制備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測序技術基礎原理課件Consequencesof2BasePairEncoding

Detectingasinglecolordoesnotindicateabase

EachreadingcontainsinformationfromtwobasesTodecodethebasesyoumustknowoneofthebasesinthesequenceACGTACGT2ndBase1stBaseConsequencesof2BasePairEnACGTACGT2ndBase1stBaseIfknowfirstbaseisanAthenimmediatelyitdecodes2ndbase.ThismustbeanAasBluetranslates2ndbaseAiffirstbaseAAACCGGTTACCAGTTGACCAGTTGAACCGGTTAACCGGTTAGCTGATCAGCTGATCAGCTGATCATCGGCTAExample:ACGTACGT2ndBase1stBaseIfknoABISOLiDSequencingbiochemistryBase/RunTime/runReadlengthDominanterrortypeEmulsionPCRSequencingbyligation50Gb>10days2*50bpSubstitutionEmulsionPCR邊連接邊測序（SBL）50G/大于10天/一個RUN讀長：50bp（可雙向）錯誤類型：替換ABISOLiDSequencingbiochemisIlluminasolexaABISOLiDRoche454IlluminasolexaGenomeSequencer20Syste（2005）GenomeSequencerFLXSyste（2006）GSFLXTitanium（2008）發展歷程：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聚合測序0.4-0.6G/10h

/一個RUN讀長：400bpMax：800bp錯誤類型：插入&缺失Roche454Sequencingbiochemis第二代測序技術小結454測序儀：

454測序儀使用的方法，經微乳液PCR發擴增后，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序，每一輪測序反應都會摻入一個核苷酸，隨后加入反應試劑熒光素和腺苷酰硫酸。這樣在每一個小孔中每當有聚合酶將核苷酸摻入到模板上都會發光。最后用腺苷三磷酸雙磷酸酶洗滌去掉多余的核苷酸。(對重復序列如polyA的測定不準確，因熒光信號具有累加效果)Solexa測序儀：Solexa測序儀使用橋式PCR直接在芯片進行模板擴增，然后同時加入四種經過修飾的脫氧核苷酸，每一個核苷酸都攜帶一種熒光集團和一個可被去除的終止基團。經過修飾的DNA聚合酶催化引物延伸測序反應。采集圖像、然后切除熒光標記基團和終止基團，重復上述反應，完成測序。（邊合成邊測序）Solid測序儀：Solid測序儀使用微乳液PCR法擴增模板片段，然后吸附有大量擴增片段的直徑1um的磁珠倍制成高密度測序芯片，借助使用連接酶而不是聚合酶測序法完成測序。在solid測序一中，每一次反應都會在引物末端加上一個熒光標記的8bp的探針，在探針中央的兩個堿基上標記有熒光基團，探針被連接上之后發出熒光，隨后熒光基團部分被切除，重新系下一輪反應。（邊連接邊測序）第二代測序技術小結454測序儀：454測序儀使用的方法，經

第一代測序技術versus

第二代測序技術第一代測序技術CurrentpopularsequencingplatformCompanyFormatReadLength(bases)讀長(bp)ExpectedThroughput

(Gb/Run)測序通量AppliedBiosystemsCapillaryelectrophoresis10003-4MSolexaParallelmicrochip75+7550SolidSequencingbyligation50+5050454LifeSciencesParallelbeadarray200(400)+200(400)0.4Currentpopularsequencingpla第三代測序技術第三代測序技術Heliscope單分子測序儀--HelicosBiosciences測序原理：邊合成邊測序特點：無需對待測模板進行擴增，采用高靈敏度的熒光探測儀，直接對單鏈的DNA模板進行合成測序，序列讀長為24到70個堿基，平均讀長為32個堿基。流程：（1）待測文庫片段化；（2）3’端加polyA尾，并與固定在芯片上的polyT進行雜交，將待測模板固定到芯片上，制成測序芯片。（3）通過DNA聚合酶將熒光標記的單核苷酸滲入到引物上，每一輪反應加入一種dNTP。（4）采集熒光信號，切除熒光標記集團，進行下一輪測序反應。Heliscope單分子測序儀測序原理：邊合成邊測序斯坦福大學的科學家最近利用HelicosBiosciences的Heliscope單分子測序儀，對一名白人男子的基因組進行了測序，文章發表在最新一期的《NatureBiotechnology》在線版上。利用一臺Heliscope測序儀和4次數據收集運行，完成了此次測序。研究人員報告稱，他們產生了數十億個Heliscope序列讀取，覆蓋了90%的人參考基因組，覆蓋度達28倍。序列讀長為24到70個堿基，平均讀長為32個堿基。到目前為止，他們已經鑒定出280萬個SNP和752個拷貝數變異。測序花了4個星期的時間，試劑花費為48000美元

斯坦福大學的科學家最近利用HelicosBiosciencSinglemoleculerealtime(SMRT)DNA測序技術—PacificBiosciences（1）以SMRT芯片載體：帶有3000個直徑為70nm左右的納米級小孔的金屬片，將DNA聚合酶、待測序列和不同熒光標記的dNTP放入到ZMW孔中，進行合成反應。（2）SMRT技術的測序速度很快，可達到每秒大約10個dNTP。、它實現了DNA聚合酶內在自身的反應速度，一秒可以測10個堿基，測序速度是化學法測序的2萬倍。（3）它實現了DNA聚合酶內在自身的processivity（延續性，也就是DNA聚合酶一次可以合成很長的片段），一個反應就可以測非常長的序列。二代測序現在可以測到上百個堿基，但是三代測序現在就可以測幾千個堿基。這為基因組的重復序列的拼接提供了非常好的條件。（4）它的精度非常高，達到99.9999%。Singlemoleculerealtime(SMRT納米孔單分子技術--OxfordNanopore公司測序原理：不同堿基產生的電信號進行測序。步驟：特殊材料制成的納米孔，孔內共價結合有分子接頭環糊精，核酸外切酶切割單鏈DNA時，被切割下來的堿基落入納米孔，并與環糊精相互作用，短暫影響流過納米孔的電流強度，電流強度的變化幅度成為每種堿基的檢測特征。堿基在納米孔中的平均停留時間是毫秒級的，一定強度的電壓可保證在電信號記錄后將堿基從納米孔中清除。獨特特點：直接讀取甲基化的胞嘧啶。納米孔單分子技術測序原理：不同堿基產生的電信號進行測序。測序技術基礎原理課件測序技術基礎羅龍海2009-03-02測序技術基礎羅龍海Sanger測序技術原理第二代測序技術原理第三代測序技術原理Sanger測序技術原理1950196019701980199020002010測序技術發展史DevelopmentofSangerSequencing(1977)InventionofAutomatedFluorescentSequencer(1985)InventionofCapillarySequencer(1996)InventionofAppliedBiosystemsSolidSystem(2007)InventionofIlluminaGenomeAnalyzerSystem(2006)Inventionof454GS20Sequencer(2005)chemicaldegradationmethodbyMaxam-Gilbertmethod(1977)ChemicaldegradationmethodbyWhitfield(1954)InventionofHeliscopesinglemolecularsequencerInventionofSinglemoleculerealtime(SMRT)DNAsequencingInventionofNanoporesinglemolecularsequencing(OxfordNanoporecorporation)1950196019701980199020002010測序測序技術基礎原理課件測序技術基礎原理課件測序技術基礎原理課件Sanger測序法原理Dr.FredSanger"dideoxy"sequencingtechnique(Sangeretal.,1977)DNA雙脫氧鏈終止法測序FrederickSangerwasawardedtheprizeinboth1958and1980.

HeisthefourthpersonintheworldtohavebeenawardedtwoNobelPrizesandtheonlypersontoreceivebothinchemistry.

Sanger測序法原理Dr.FredSanger"d測序技術基礎原理課件測序技術基礎原理課件測序技術基礎原理課件IlluminaGenomeAnalyzerABISOLiDRoche454第二代測序技術IlluminaGenomeAnalyzer第二代測二代測序技術200520062007年份原理Pyrosequencing邊合成邊測序邊連接邊測序454SolexaSOLID二代測序技術200520062007年份原理PyrosequIlluminaSolexaABISOLiDRoche454第二代測序技術IlluminaSolexa第二代測序技術可逆阻斷技術可逆阻斷技術IlluminaSolexaFlowcellFlowcell一個flowcell

包括8個lanes

Lane1Lane8Eachlanecontainsmultipletiles–total100每個lane上有許多個tiles——共計100個（見上圖）Eachtileisimagedfourtimespercycle–oneimageperbase每個循環會對每個tile照4次相——每個堿基都會成像Imagefrom1tileIlluminaSolexaFlowcellFlowcGenomeAnalyzerAnalysisPipelineLibraryPreparationClusterStationGenomicmRNASmallRNAChIP-SeqIllumina/GAWorkflow

工作流程*GrowClusters*5hours*Orsplitprocessinstages*Safestoppingpoints*StartSequencing*ClusterDensityEvaluation*4imagespertilepercycle*Runtime:2-3days*Firecrest:ImageAnalysis*Bustard:Basecalling*Gerald:SequenceAlignment文庫構建Cluster工作站基因組測序分析*生成“簇”*5小時*開始測序*序列簇的豐度檢測*一個循環每個tile照4張照片*2-3天*圖像分析*Bustard:basecalling*Gerald:序列分析?樣品預處理PCR成簇測序拼接分析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）（純化連接產物）（）（）（）（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片段的文庫構建27bp27bp27bp27bpGenomicDNAFrClusterGeneration序列簇的產生PrepareDNAfragmentsLigateadaptersAttachsinglemoleculestosurfaceAmplifytoformclustersRandomarrayofclusters（Cluster的隨機排列）

100um~1000moleculesper~1umcluster

~20.000clusterspertile32-40millionclustersperexperiment

（1個cluster上有1000個分子

1個tile上有20,000個cluster

每個實驗可完成3.2-4千萬個cluster)通過cluster將單分子放大、固定ClusterGeneration序列簇的產生PreparOHOHGraftedflowcelldiol

P7

P5

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

G

T…T

G

C

T

A

C

G

A

T…TheidentityofeachbaseofaclusterisreadofffromsequentialimagesBaseCalling123789456TTTTTPairedEndPairedEndPairedEnd雙末端測序、正反雙向測序SamplePreparation樣品前準備ClusterGeneration分子簇的生成SequenceBySynthesis邊合成邊測序（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)

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

U-P5

OHOHU8oxo-G?OHOHdiolP7P5GrafteTemplatehybridizationUUOHOHGraftedflowcellU

P7

P5ClusterGeneration:InitialExtensionInitialextensionUU1stcycleDenaturationUU1stcycleannealingUU1stcycleextensionUU2ndcycledenaturationUU2ndcycleannealingUUUTemplateUUOHOHGraftedflowcen=25totalClusterGeneration:Amplification成簇2ndcycleextensionUUUClusterAmplificationUUP5Linearization(USER)胞嘧啶識別位點處切割BlockwithddNTPs末端ddNTPs封閉DenaturationandHybridizationSBS325個循環n=25ClusterGeneration:AmplifSequencing測序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測序DenaturationandSequencingbiochemistryBase/RunTime/runReadlengthDominanterrortypeDNAcluster、Reversibleterminators、SequencingbySynthesis40-50Gb10days2*75bpSubstitutionIlluminaSolexa形成DNA簇；可逆阻斷技術邊合成邊測序（SBS）40-50G/10天/一個RUN讀長：75bp（可雙向）錯誤類型：替換SequencingbiochemistryBase/RIlluminasolexaABISOLiDRoche454IlluminasolexaSOLID測序技術SOLID測序技術AB/SOLIDWorkflow工作流程文庫制備EmulsionPCRBeadsEnrichment微珠沉積連接測序數據分析AB/SOLIDWorkflow工作流程文庫制備文庫制備EmulsionPCRBeadsEnrichment微珠沉積連接測序數據分析WorkFlow：文庫制備WorkFlow：序列可以用超聲波、機械剪切或酶解等方法，隨機或者定向的打斷成小片段序列可以用超聲波、機械剪切或酶解等方法，隨機或者定向的打斷成（大片段兩頭測序）“Mate-paired”步驟：環化——切割——加接頭——測序（大片段兩頭測序）“Mate-paired”步驟：環化——酶切為粘性末端對于復雜的分子環化，采用低濃度的模板濃度進行連接酶切為粘性末端對于復雜的分子環化，采用測序技術基礎原理課件文庫制備EmulsionPCR微珠富集微珠沉積連接測序數據分析WorkFlow：文庫制備WorkFlow：2.EmulsionPCR+TemplatesEnzyme+dNTPsP1-coupledDNAbeads~100,000P1sitesperbeadStartwith2BillionbeadsperemulsionPolymerase100,000P1位點/每個bead2Billionbeads/每個emulsion2.EmulsionPCR+TemplatesEnzMixPCRaqueousphaseintoawater-in-oilemulsionandcarryoutemulsionPCRReactorwithtemplate,beadandPCRreagentsMineraloil+surfactantsMixPCRaqueousphaseintoawBeadscollectedfollowingemulsionPCR:Beadswithamplifiedproduct(~40KPCRproductsperbead)BeadswithnoproductP1P2P2Beadscollectedfollowingemul文庫制備EmulsionPCR微珠富集微珠沉積連接測序數據分析WorkFlow：文庫制備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文庫制備EmulsionPCR微珠富集微珠沉積連接測序數據分析WorkFlow：文庫制備WorkFlow：4.Depositebeads3’-endmodificationBeadsattachedtoglasssurfaceinarandomarrayTemplatebeaddeposition4.Depositebeads3’-endmodi文庫制備EmulsionPCR微珠富集微珠沉積連接測序數據分析WorkFlow：文庫制備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測序技術基礎原理課件Consequencesof2BasePairEncoding

Detectingasinglecolordoesnotindicateabase

EachreadingcontainsinformationfromtwobasesTodecodethebasesyoumustknowoneofthebasesinthesequenceACGTACGT2ndBase1stBaseConsequencesof2BasePairEnACGTACGT2ndBase1stBaseIfknowfirstbaseisanAthenimmediatelyitdecodes2ndbase.ThismustbeanAasBluetranslates2ndbaseAiffirstbaseAAACCGGTTACCAGTTGACCAGTTGAACCGGTTAACCGGTTAGCTGATCAGCTGATCAGCTGATCATCGGCTAExample:ACGTACGT2ndBase1stBaseIfknoABISOLiDSequencingbiochemistryBase/RunTime/runReadlengthDominanterrortypeEmulsionPCRSequencingbyligation50Gb>10days2*50bpSubstitutionEmulsionPCR邊連接邊測序（SBL）50G/大于10天/一個RUN讀長：50bp（可雙向）錯誤類型：替換ABISOLiDSequencingbiochemisIlluminasolexaABISOLiDRoche454IlluminasolexaGenomeSequencer20Syste（2005）GenomeSequencerFLXSyste（2006）GSFLXTitanium（2008）發展歷程：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