Illumina測序原理課件

Illumina測序原理目錄概覽制備文庫(LibraryPreparation)在測序芯片上生成DNA簇(ClusterGeneration)邊合成邊測序(SequencingbySynthesis)雙側(cè)測序，配對測序(Paired-End,MatePair&Multiplexing)數(shù)據(jù)分析(DataAnalysis)LibraryPreparation制備文庫Sequencing測序

DataAnalysis數(shù)據(jù)分析43ClusterGeneration生成DNA簇2測序工作流程概覽1DNA

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LibrarypreparationClustergeneration5’5’3’GTCAGTCAGTCACAGTCATCACCTAGCGTAGT123789456ImageacquisitionSequencing測序過程概覽SequencingOverviewBasecallingT

G

C

T

A

C

G

A

T…制備文庫

LibraryPreparation

LibraryPrepOverview(gDNA)DNATemplate1-5μg，50μlpurifiedDNAOD260/280=1.8-2.0AsintactaspossibleLibraryYield500-1000ngOD260/280=~1.8SR:150-250bpPE:300-650bpMW=sizeinbpx650DNA片段化(Fragmentation)我們用的片段化方法CovarisBioruptor別的可選的片段化方法NebulizerSonicationHydroShear?EnzymaticOthers打碎Covaris修補(bǔ)末端T4,Klenow,T4PNK加A堿基AAAAAAKlenowexo-PPAA5’5’5’3’5’T3’TAAP3’5’T5’3’TT3’5’TA3’5’2%AgaroseGel電泳切膠選取DNA，對于單側(cè)測序選取150-250bp，對于雙側(cè)測序選取600bp磁珠或Qiagen等回收試劑盒回收DNA片段AdaptorsLigation&ExcisionPCR擴(kuò)增5’T3’A5’AT3’富集5’T3’A5’AT3’退火5’T3’A5’AT3’3’A5’T3’TA5’延伸5’3’5’T3’TAA10-12CyclesLibraryQC雙側(cè)測序:500bp+100bp單側(cè)測序:250bp+100bpLibraryPreparationSummaryRepairEndsAddanAtothe3’EndsFragmentDNASizeSelectonGelLigateAdaptersPCRFragments<800bpBluntEndFragmentswith5’–Phosphorylatedends3’-dAOverhangAdapter-ModifiedEnds300-600bpFragmentsAmplifiedDNAwithAdaptersGenomicDNALibraryPurifiedGenomicDNAQCLibrary

FASTLIBRARYPREPIN

LESSTHAN2HOURSClosedtubeDNAfragmentationTransposon-mediatedlibrarypreparationUltra-lowinputrequirements(50ng)ENABLESARANGEOFCEANDNGSAPPLICATIONSEpicentreNexteraTechnology

SingleTube,RapidLibraryPrep生成DNA簇

ClusterGeneration

測序芯片(Flowcell)生成DNA簇(ClusterGeneration)每個簇有~1000-6000個DNA分子OHOHGraftedflowcell種有引物的芯片表面diol

P7

P5TemplateHybridizationdioldiolTemplateHybridization模板雜交dioldiolInitialextension啟始延伸dioldiol

Denaturation解鏈1stcycle

annealing第一次退火dioldioln=25/28total 1stcycle

denaturation第一次變性dioldiol1stcycle

extension第一次延伸dioldioldioldiol2ndcycle

denaturation第二次變性2ndcycle

annealing第二次退炎dioldioldiolBridgeAmplificationdioldioldiol2ndcycle

extension第二次延伸循環(huán)25~28次Lin_Blocking_PrimerHybClusterAmplification擴(kuò)增生成DNA簇OHdioldiolOHLinearization單鏈化OHBlockingwithddNTP(

)堵上3‘端DenatureandHybridization變性再雜交OH邊合成邊測序

SequencingbySynthesisCAGTCATCACCTAGCGTA5’GTCAGTCAGTCAGT3’5’

第一個堿基結(jié)合(Firstbaseincorporated)Cycle1:

檢測光信息(DetectSignal)切掉終止子和染料(CleaveTerminatorandDye)Cycle2-n:循環(huán)(Repeat)SequencingbySynthesisACGTDataAnalysis123789456TTTTTTT

G

T…T

G

C

T

A

C

G

A

T…SingleReadSequencingSummaryDataAnalysisImageAnalysisBaseCallingAssemblyClusterGenerationTemplateHybBridgeAmplificationLin_Block_HybSequencingIncorporationImage-takingCleavingLibraryPreparationFrag_Repair_AddAAdaptorLig_ExcPCR_QC單側(cè)測序、雙側(cè)測序、配對測序、多重測序

SR,PE,MP,Multiplexing單側(cè)測序

SingleReadSequencingSequencereadsKnownsequenceNewsequence80-90%兩側(cè)測序

Paired-End

SequencingSequencereadsKnownsequenceNewsequence95to>99%OHOHdiol

P7

P5

兩種植有引物的測序芯片GraftedFlowCells:SRvsPE8oxoG-P7

U-P5

OHOHU8oxo-GSingleReadPeriodateLinearization單側(cè)測序芯片一種引物有修飾PairedEndUracilSpecificExcisionReagent(USER)formamidopyrimidineglycosylase(fpg)雙側(cè)測序芯片兩種引物都有修飾

DenaturationandHybridization變性和雜交DenaturationandDe-Phosphorylation(PNK)變性，去掉磷酸基，活化另一側(cè)引物OHOHResynthesisofP5Strand再次合成第二鏈OHP7Linearization(fpg)打斷第一鏈OHBlockwithddNTPs堵上3’端DenaturationandHybridization變性再雜交Read2:Resynthesis&SBSSequencing第一鏈測序Read1Sequencing第二鏈側(cè)序Read2(Read1) 讀第一鏈(Read2) 讀Index(也稱作Barcode，6bptag)(Read3) 讀第二鏈123Multiplexing配對測序

Mate-PairSequencing2kb–10kbBBBBBPaired-EndSequencing數(shù)據(jù)分析

DataAnalysis數(shù)據(jù)分析流程

DataAnalysisWorkflowSYSTEMCONTROLSOFTWAREReadGenerationCASAVAAlignments,variations,buildsVISUALIZATIONGenomeStudio,orfavoritebrowserPrimaryAnalysisHCS 儀器運(yùn)轉(zhuǎn)；收集信號RTA 實(shí)時圖片分析；堿基識別；質(zhì)量評估OLB 離線圖片分析SecondaryAnalysisCASAVA 片段組裝，序列比較， SNP識別，indel識別，mRNA/miRNA定量VisualizationGenomeStudio 數(shù)據(jù)的直觀展示IlluminaDataAnalysisToolsRTA=OLBHCSRTAbasecallsqualityscoresreadssummariesallelesSNP’sindelscounts(RNA)GenomeStudioOLBimagesbasecallsqualityscores(optional)CASAVACASAVA+HCSRTA=(SCS/RTA)+OLB+CASAVA(resultsusingonlySCS/RTAandCASAVAarethesameasacomboofRTA,OLBandCASAVA)CASAVA分組：Separatingthemultiplexedsamplesbeforealignment.組裝：Alignment(Gerald)byELANDv2Gappedalignments(upto20bases).Multiseed(maximum4).多態(tài)：TheSNPcaller突變：Theindelfinder(onlyonpairedendruns)Reportsinsertionsanddeletionsuptohalfthereadsize.定量：mRNA-seqcountingNGSSequencingData

ASCIICharacterQ-scorePF(0,1)SequenceInstrumentRunIDLaneTileX-coordY-coordIndex#Read#數(shù)據(jù)質(zhì)量值Quali