高通量測序技術2017年3月21號illumma9旬亟＞appliedbiosystemsThermoFisherSCIENTIFICMeItechnologies,主要內容、高通量測序簡介Solexa和Semiconductor測序原理三、高通量測序部分應用、高通量測序簡介■高通量測序(High?throughputsequencing)又稱"下一代”測序(aNext-generation?sequencing),可一次性對幾百萬到十億條DNA分子進行并行測序；■可以對一個物種的基因組和轉錄組進行深入、細致和全貌的分析，所以乂被稱為深度測序(Deepsequencing)。高通量測序的發(fā)展o1992年LynxTherapeuticsMPSSo2003年PolonySequencing（哈佛）o2005年454Pyrosequencingo2006年SolexaSequencing-By-Synthesiso2007年ABISOLIDo2008年HelicostSMSSequencingo2009年IonTorrentSemiconductorSequencingo2011^PicficBiosciencesSMRTSequencing高通量測序的主要代表平臺有：羅氏的(Roche)的454測序儀(RocheGSFLXsequencer);Illumina的Solexa基因組分析儀(IlluminaGenomeAnalyzer); 丿ABI的SOLiD測序儀(ABISOLiDsequencer);ThermoFisher的離子半導體測序儀(IonSemiconductor^sequencer).

二、Solexa和Semiconductor測序原理Illuminasolexa文庫制備A>Ai.FragmentgenomicDNAAn.Double-strandedcDNA

(fromfiqure2B)A>Ai.FragmentgenomicDNAAn.Double-strandedcDNA

(fromfiqure2B)B.EndrepairandphosphorylateD.LigateindexadapterP5Rd1SPDNAInsertIndexA.>ARd2SP'P7'P5Rd1SPDNAInsertIndexA.>ARd2SP'P7'E.DenatureandamplifyforfinalproductC.A-tailingFlowcell□□□□口口口口口口口口口口口口口口口口口口口口口口口□□□+"□□3明口口口口口口口口口口口口口口口口口口口田口口口口41TileEachcolumncontains60tilesEachtileisimaged4timespercycle簇生成，模板變性OHP7flowcellTemplate

hybridizationInitialextensionDenaturation橋式擴增1st1stcycle

denaturation1stcycleannealing1stcycle 2ndcycleextension denaturation2ndcycleextension2ndcycleannealingNextCycleNextCycle并行合成測序法(SBS,Sequencing-By-Synthesis)和可逆性末端終止技術(ReversibleTerminatorChemistry)qCycle1:AddsequencingreagentsqFirstbaseincorporatedDetectSignalCleaveTerminatorandDyeCycle2-n:Addsequencingreagents

andrepeat

信號收集Clustersarebrightspotson

animageEachclusterrepresents

thousandsofcopiesofthe

sameDNAstrandina1-2

micronspot讀取堿基序列在測序過程中，每種堿基被激發(fā)后都會發(fā)出特殊波長的熒光在每個循環(huán)過程中每一個簇只對應一張圖像■嚙襲■■DOBTA3AT WWW.TTTTTTTGTIllumina測序平臺展示MiSeqMiSeqNextSeq500測序通量讀長測序時長數(shù)據(jù)量2xl50bp2x75bp~29h18h100-200GB50-80GB1x75bpUh25-30GB適用范圍全基因組測序、全外顯子測序、轉錄組測序特點高通量、靈活性、簡便讀長測序時長數(shù)據(jù)量2x75bp~24h3.3-3.8GB測序通量2xl50bp~24h4.5-5.1GB2x250bp~39h7.8-8.5GB2x300bp~55h13.2-15GB適用范圍靶向測序、/」\基因組測序、靶向基因表達分析特點贏薩單次運行產生XOObP雙部缽Hiseq4000測序通量讀長測序時長 數(shù)據(jù)量2xl50bp1300-1500GB1x75bpl-3.5d 650-750GB1x50bp215-250GB適用范圍全基因組測序、全夕卜顯子測序、轉錄組測序特點最高的通量，每個樣品的最低價格讀長測序時長數(shù)據(jù)量測序通量2xl50bp<3d單流動槽 雙流動槽8-9TB 16-18TB適用范圍全基因組測序特點超高的通量，有望突破人類全基因組測序的$1000大美HiSeqXTen流動槽察S1S2S3S4lx50bp167GB280-333GBN/AbN/Ab<19h數(shù)據(jù)量a1x75bp333GB560-667GBN/AbN/Ab<29h2x150bp500GB850-1000GB2000GB3000GB<40h適用范圍全基因組測序、臏重測序、轉錄組測序特點對幾乎任何基因組、任何測序方法和規(guī)模的項目,量，靈活簡便。都有可擴展的通所有產出和read數(shù)量規(guī)格都基于單個流動槽計算，NovaSeq6000系統(tǒng)可同時運行1個或2個流動槽。N/A:不適用。NovaSeq6000

IonSemiconductor測序流程Long/MidRangeAmp. 、BarcodedLibraryCreationLRFragmentsBarcodedLibraryLibraryPreparation； BCAmplicon P1SequenciSequenciClonalAmplificationontoIonSphereParticles(ISPs)IsolatePositiveISPs?LoadPGM?ChipandRunPGM?Sequencer'、、 ?TorrentServerCollectsDataDuringSeq.Run?AnalyzewithTypeStreamPlug-In文庫制備LibraryProparationBindngSeparationDNAQuantification:QuantificationofeachsampleisperformedusingtheQubit?InstrumenttopreparefordownstreamstepsoftheworkflowEthinolWnhButionBufferTrantferLibraryProparationBindngSeparationDNAQuantification:QuantificationofeachsampleisperformedusingtheQubit?InstrumenttopreparefordownstreamstepsoftheworkflowEthinolWnhButionBufferTrantferPCR產物純化Purification:?RemovalofexcessPCRcomponentswiththegoalofisolatingthePCRproducts

usingAMPure?reagentfromBeckman-Coulter￡AjercourtAMF\ireXP接頭連接Fragmentation:AmpliconPCRProductsPre-FragmentabonAAdapterAmplicon:Fragmentation:AmpliconPCRProductsPre-FragmentabonAAdapterAmplicon:OutputofFragmentationreactionA-Adapter:ComplementarytoasequencingprimerAdaptorLigation:BarcodeP1AdapterBarcodeP1AdapterBarcode:Usedtotag/identifyaspecificDNAsampleP1Adapter:FacilitatesattachmenttotheIonSphere?Particles(ISPs)PCRProductsPost-FragmentationBarcodedLibraryBCPI片段篩選?SizeselectionisaccomplishedthroughtheuseofAMPure?reagentfromBeckman-

CoulterAlteringthebead:sampleratioallowsforselectionofthetargetsizedistribution樣本混池QuantificationAllsamplesarequantifiedonefinaltimetoensureequalrepresentationusingtheAgilent.TapeStationInstrument:PoolingSample4BCAmpliconP1Sample4BCAmpliconP1克隆擴增Ibmplat?

PreparationIdealClonalAmplification:1IonSphere?Particle(ISP)+1BarcodedLibraryFragmentIsothermalAmplificatbn1IonSphere?Particle(ISP)+ClonallyAmplifiedBarcodedLibraryFragmentsAmplification不理想的擴增情況TemplatePreparationISP富集(IonSphereParticalEnrichment)EnrichmentStep1AddMagnetic

StreptavidinBeadEnrichmentStep2:ImmobilizetoMagnetandWashEnrichmentStep3:

DenatureISP

withNaOHTemplatePreparationReadsof

Interest

(1library

fragment/ISP)Polyclonal

ReadsProductof

ISPEnrichmentNon-

templated

ISP擴增引物的5'端標記了生物素,通過鏈霉親和素標記的磁珠與標記了生物素的DNA結合.

測序4dNTPs自然情況下，DNA聚合酶將一個核昔酸滲入到DNA分子中就會釋放出一個H+,導致局部可檢驗的PH值發(fā)生變化，被離子傳感器檢測到，從而轉換為數(shù)字信號。堿基識別依次摻入4中堿基連續(xù)兩個相同堿基結合IonTorrent儀器展示Ion314?Chip1millionwellsIon316?Chip6millionwellsIon318?Chip11millionwells1-2samples<24samples<48samplesIonTorrentPGMChip建PGM314PGM316PGM318傳感器數(shù)1.3M6.3MUM| 數(shù)據(jù)量~100MB~1GB-2GB運行時間2.3/4.7h3.0/4.9h4.4/7.3h讀長200/400bp2OO/4OObp2OO/4OObp應用氾圍擴增子測序、靶向重測序、小基因組測序特點基于半導體技術測序I纏，快速“讀"出堿DNA序列人基因組富高通量測序I湖全基因組測序動植物基因微生物基因咼通量測序（DNA外顯子組測序甲基化測序擴增子測序向捕獲測序全外顯子纟ImRNA測序LncRNA測序RNA SmaHRNA測序CircularRNA測序全基因組測序?全基因組重測序是對已知基因組序列的物種進行不同個體的基因組測序，通過序列比對，可以找到大量的單核昔酸多態(tài)性位點(SNP),插入缺失位點(InDei)、結構變異位點(SV)位點和拷貝數(shù)變異位點(CNV)。?全基因組denovo測序是指對未知基因組序列的物種進行從頭測序，用生物信息學分析方法進行拼接、組裝，從而獲得該物種的基因組序列圖譜。pawed，EZJdGene&GcFsarsMaiemnd刀。gemrglCFoFsnrssenucngpaireQEnQ外顯子組測序O外顯子組蟲一個物種基因組中全部外顯子區(qū)域的總和；+人外顯子組只占基因組的?1%,約30Mb；4約85%的人類致病突變都位于這1%的蛋白質編碼序列上。Day1OlagmentGenomicDNAReagents:50nggChW.ST2,FD,TDE2,Tris-HC110mM.pH85?CleanUpTagmenteoDNAReagents:RSB.SP3.Fresh80%EtOH文庫構建AmplifyTagmentedDNAReagents:Irxlex1.Ind^x2.LAMSateStoppingPolniCleanupAmplifiedDNAReagents:RSB,SP3,Fresh60%EtOHSafeStoppingPoint[Recommended]Day2?HybridizeProbesRaagents:BLR.CEMEX,EHB1,EHB2,RSB,SPBOCaptureHybridizedProbesReagents：EE1,Er2,EEW,HP3.RSB.SMBSafeStoppingFConstructshotgunlibroryt■HybridizationGenomicDNA FragmentsCPerformSecondHybridizationWFReagents:BLR.CEX/EEX.EHB1.EHB2.SPB?PerformSecondCaptureRoagants:EE1,ET2.EEW,HP3SMRMapping,alignment,variantcallingAGGTCGTTACGTACGCTAC□ACCTACATCAGTACATAG<—GCATGACAAAGCTAC?TGTCleanUpCapturedLibraryRcagcntaRSB.SP9.Fresh60%EtOHSateStoppingPointOptiofialCXremiSafeStoppingPointNimblegen、AgilentAmplifyEnrichedLibraryReagouls:EAM,PPGCleanUpAmplifiedEnrichedLibraryReagents:RSB,SP9,Fresh80%EtOH(■Pre-Amp?Post-AmpIlluminamRNA測序omRNA測序(mRNAseq)是指利用高通量測序技術對cDNA進行測序從而得到一個樣品中基因表達、cSNP、全新的轉錄、全新異構體、剪接位點、等位基因特異性表達和罕見轉錄等最全面的轉錄組信息。文庫構建鏈特異性試劑盒建庫 通過帶oligoT的磁珠分離出mRNA度性RNA RNA片段 cDNA第一鏈 cDNA第二鏈 打斷反轉錄dTTPBgy^dUTPcDNA第二鏈合成 加接頭UNG覲理 擴增文庫LncRNA(longnoncodingRNA)測序oLncRNA是一類長度超過200nt的長鏈非編碼RNA,通過與DNA、RNA或蛋白質結合，在表觀遺傳、轉錄及轉錄后等水平調控基因表達。oIncRNA測序是研究已有參考基因組物種的特定組織或細胞在某個特定時期轉錄出的所有IncRNA和mRNAo文庫構建gA提取針對不同的雜樣品制定不同的提取方案總RN.A質盡檢則 ?濃度及純度槍測誕性棄用田3專用席脂桶也冰女是入曲in21WBBjyzer險測rRNA去除通jgA去除試利盒去除收RNA片段化通過圈子打斷的方式，播RNA打0F卻統(tǒng)-粕此p片段通溯機引歯和逆轉錄髄作用合芯D\?A第一犠r臺覦DM第二継時，注

錐特肄性發(fā),將dggia^dUTP,大取提高結果的g性H大場3<iCH0bbpAstern21corm實2W槍測立旌大小,