RNA-seq研究方法与策略

.,RNA-seq研究方法与策略,市场部张壮壮zhangzz上海天昊生物科技有限公司,.,DNAmakesRNAmakesprotein,mRNA是沟通DNA和蛋白质的“桥梁”,.,MessengerRNA(mRNA)isalargefamilyofRNAmoleculesthatconveygeneticinformationfromDNAtotheribosome,wheretheyspecifytheaminoacidsequenceoftheproteinproductsofgeneexpression.Anon-codingRNA(ncRNA)isafunctionalRNAmoleculethatisnottranslatedintoaprotein.,microRNAs(miRNAs)Smallnon-codingRNAsof22nucleotidesthatareintegralcomponentsofRNA-inducedsilencingcomplex(RISC)andthatrecognizepartiallycomplementarytargetmRNAstoinducetranslationalrepression,whichisoftenlinkedtodegradation.Longnon-codingRNAs(longncRNAs,lncRNA)arenon-proteincodingtranscriptslongerthan200nucleotides.,ChrisP.Ponting,PeterL.Oliver,andWolfReik.EvolutionandFunctionsofLongNoncodingRNAs.Cell136,629641,February20,2009.,RNAworldismorecolorful,.,DualRNA-seqofpathogenandhost.10,618630(2012).,RNAType,.,一个典型的快速生长的哺乳动物细胞培养中,每个细胞大约含有10-30pg的RNA，而一个完全分化的原代细胞中，RNA的量要少得多大约每个细胞中RNA的含量小于1pg。细胞中的RNA分子主要是tRNA和rRNA。mRNA大约占细胞中RNA总量的1-5%，但是具体的量取决于细胞类型和细胞的生理状态。,.,RNA的特点,分子相对较小，通常是单链；周期短，降解快；通常有特殊结构(mRNA、miRNA、tRNA和rRNA)；通常有前体，需要剪切和修饰(mRNA、miRNA、tRNA和rRNA)；,mRNA的特点,5端帽子结构和3端PolyA尾巴分子长度一般介于500-10000nt有前体，包含内含子能翻译成功能蛋白,原核生物mRNA缺少cap和Poly-Atail的结构!,.,一个动物细胞中，大约有360,000个mRNA分子，组成了大约12,000个转录本，一个典型转录本的长度大约为2kb。一些mRNA分子占到了总mRNA的3%，而其它的mRNA分子的含量低于0.01%。这些“稀有的”或者“低丰度”的mRNA分子在每个细胞中只有5-15个拷贝。但是，这些稀有的mRNA大约有11,000种，占到了mRNA数量的45%。,Challenge,.,TranscriptomeThetranscriptomeisthecompletesetoftranscriptsinacell,andtheirquantity,foraspecificdevelopmentalstageorphysiologicalcondition.Tocatalogueallspeciesoftranscript(mRNAs,ncRNAs);Todeterminethetranscriptionalstructureofgenes(theirstartsites,5and3ends,splicingpatternsandotherposttranscriptionalmodifications);Toquantifythechangingexpressionlevelsofeachtranscriptduringdevelopmentandunderdifferentconditions.,RNAsequencingRNA-seq(RNASequencing),alsocalled“WholeTranscriptomeShotgunSequencing”(“WTSS”),isatechnologythatutilizesthecapabilitiesofnextgenerationsequencingtorevealasnapshotofRNApresenceandquantityfromagenomeatagivenmomentintime.,.,但通常我们使用的是RNA-seq的狭义概念，亦即mRNA-seq。,NecsuleaA,KaessmannH.Evolutionarydynamicsofcodingandnon-codingtranscriptomes.NatRevGenet.2014Nov;15(11):734-48.,.,Trendsin“TranscriptomeandRNA-seq”,.,.,GENReports:Market某些特殊分子特征只能在RNA水平才能观察到可变剪接、基因融合、RNA编辑等;Keymutation对mRNA转录本表达量的影响如剪接位点、motif等位置的突变;,WhyweneedRNA/RNA-seqstudies?,First,Second,.,直接得到核酸序列信息，除了得到基因表达量的差异，更可以检测RNA的结构和结构变异。开放性的转录组分析：无需参考基因组信息，无需设计探针，不但能检测已知基因还能够发现新的转录本。在测序覆盖率足够大时能够检测到细胞中的低丰度转录本。随着测序深度的增加可以获得更广的动态检测范围，能够同时鉴定和定量高丰度转录本和低丰度转录本。价格相对便宜,RNA-seqConclusion,Third,.,.,2.RNA的提取与质检,3.测序文库的构建,4.上机测序与数据质控,5.数据分析与结果展示,1.试验方案设计,.,Figure1RNA-seqworkflow.SchematicdiagramofRNA-seqlibraryconstruction.TotalRNAisextractedfrom300,000cellsto3millioncells,andasmallaliquotisusedtomeasuretheintegrityoftheRNA.rRNAisthendepletedthroughoneofseveralmethodstoenrichsubpopulationofRNAmolecules,suchasmRNAorsmallRNA.mRNAisfragmentedintoauniformsizedistributionandthefragmentsizecanbemonitoredbyRNAgelelectrophoresisorAgilentBioanalyzer.ThecDNAisthenbuiltintoalibrary.ThesizedistributionpatternofthelibrarycanbecheckedbyAgilentBioanalyzer;thisinformationisimportantforRNA-seqdataanalysis.Mappingprogramsalignreadstothereferencegenomeandmapsplicejunctions.GeneexpressioncanbequantifiedasabsolutereadcountsornormalizedvaluessuchasRPKM.IfRNA-seqdatasetsaredeepenoughandthereadsarelongenoughtomapenoughsplicejunctions,themappedreadscanbeassembledintotranscripts.Thesequencesofthereadscanbeminedbycomparingthetranscriptomereadswiththereferencegenometoidentifynucleotidevariantsthatareeithergenomicvariants(forexample,SNPs)orcandidatesforRNAediting.,RNA-seqWorkflow,Technicalconsiderationsforfunctionalsequencingassays.13,802807(2012).,?,.,Wecarriedoutreplicateexperimentsacross15laboratorysitesusingreferenceRNAstandardstotestfourprotocols(poly-A-selected,ribodepleted,size-selectedanddegraded)onfivesequencingplatforms(IlluminaHiSeq,LifeTechnologiesPGMandProton,PacificBiosciencesRSandRoche454).Theresultsshowhighintraplatform(SpearmanrankR0.86)andinter-platform(R0.83)concordanceforexpressionmeasuresacrossthedeep-countplatforms,buthighlyvariableefficiencyandcostforsplicejunctionandvariantdetectionbetweenallplatforms.,.,ForintactRNA,geneexpressionprofilesfromrRNA-depletionandpoly-Aenrichmentaresimilar.Inaddition,rRNAdepletionenableseffectiveanalysisofdegradedRNAsamples.,.,.,重复的设置：技术重复、生物学重复,技术误差和个体差异可以通过设置重复进行评估，但不能消除。只有准确平衡了技术误差和个体差异，才能用RNA-seq结果解释组间差异。,.,RNA-seq文库构建和测序的技术重复性皆为0.99以上，可以不设技术重复。,.,RNAPreparation,IsolateandpurifyRNASolubilizationMechanicalhomogenizationRecoveryofRNAfromlysate:Organicextraction/Solid-phaseextractionQuantitationandQualityAssessmentTargetenrichment：ThefourmethodsthatarecommonlyusedtoenrichspecificclassesofRNAsare:SelectionoftargetRNAsviahybridization.Removalofnon-targetRNAsviahybridization.Copy-numbernormalizationviaduplex-specificnucleasedigestion.Targetenrichmentviasize-selectionRNAfragmentation,RNAenrichmentmethodsPoly(A)-RNAselection-byhybridizationtooligo-dTbeads-maturemRNAhighlyenriched-efficientforquantitationofgeneexpressionlevel-limitation:3biascorrelatingwithRNAdegradationrRNAdepletion:-byhybridizationtobead-boundrRNAprobes-rRNAsequence-dependentandspecies-specific-commercialkits:InvitrogenRibo-minuskit;EpicenterRibo-Zerokit-allnon-rRNAretained:pre-maturemRNA,longnon-codingRNA-necessaryforprokaryoticorganismsSmallRNAextraction:-specifickitsrequiredtoretainsmallRNA:AmbionmirVanakit-optionalfinesize-selectionbygel.,.,.,ExamplesofgoodandpoorqualityRNAprepsareshowninFigureA(agarosegel)andFigureB(Bioanalyzertrace).,RNA的操作本就是项复杂、精细的工作！,RNA质量要求：,TotalRNA，溶解在H2O或TE(pH8.0)中;OD260/280值应在1.82.2之间，RNA28S:18S1.5，推荐RIN7；无DNA污染;最低浓度不低于100ng/L;每个样品总量不少于5g;,A,B,.,PrepareLibraries,First-strandsynthesis(Reversetranscriptases,)Usingoligo-dTtoprimeoffofthepoly-AtailofmaturemRNA.UsingrandomprimerstoprimeatrandompositionsalongtheRNAmolecule.PrimingoffofoligosthatareligatedontotheendsoftheRNA.Second-strandsynthesis(DNApolymerase)SynthesisbyRNAnickinganddisplacement.Usinganoligothatiscomplementarytoanadapterpre-ligatedtothe5-endoftheRNAtemplate.Usingaprimercontaininga3-oligo-dG(thismethod,referredtoasSMART)takesadvantageofthephenomenonthattheMMLVreverse-transcriptaseleavesaterminalnon-templatepoly-dC3-overhang).FragmentationofcDNASequencingadaptersRegardlessoftheplatform,twotypesofsequenceelementsarerequired:(1)Terminalplatform-dependentsequencesthatarerequiredforclonalamplificationandattachmenttothesequencingsupport.(2)Sequencesforprimingthesequencingreaction.Additionofadapters(RT/PCR,ligation)PreparationofstrandedlibrariesValidationandQuantification,.,Table3.1Listoffunctionalelementscontainedinsequencingadapters.,Commercialkits,.,Sequencing,ChoosingasequencingplatformSamplepreparationandsubmission,Furthermore,thefacilityneedstoknow:Thesequenceofthesequence-primingsite.Thelengthofthereadyoudesire.Whetheryouwantsingle-endorpaired-endreads.Whetherthereisabarcodeorindexsequence.IfusingIlluminasequencingthefacilityalsoneedstobenotifiediftheinsertscontainaregionoflowsequencecomplexityimmediatelyafterthesequence-primingsite(i.e.abarcode).,Somegeneralissuesthatneedtobeconsideredare:Thatthesamplesarecleanandfreeofmajorcontaminants.TheprimaryDNAmoleculescontaininsertsofthecorrectsize.TheprimaryDNAmoleculeshaveadaptersoneachend.Thesampleconcentrationisappropriate.Thesamplesaresuspendedinappropriatebuffers.,.,测序长度,测序数据,.,Analysis,StereotypicalRNA-seqAnalysisPipelineDemultiplex,filter,andtrimsequencingreads.Normalizesequencingreads(ifperformingdenovoassembly).denovoassemblyoftranscripts(ifareferencegenomeisnotavailable).Map(align)sequencingreadstoreferencegenomeortranscriptome.Annotatetranscriptsassembledortowhichreadshavebeenmapped.Countmappedreadstoestimatetranscriptabundance.Performstatisticalanalysistoidentifydifferentialexpression(ordifferentialsplicing)amongsamplesortreatments.Performmultivariatestatisticalanalysis/visualizationtoassesstranscriptome-widedifferencesamongsamples.,.,TotalRNA,oligodT磁珠富集mRNA,打断、双链cDNA合成,末端修复、加A加接头,片段选择,PCR扩增、纯化,rRNA去除,文库质量检测,Illumina测序,片段大小筛选,oligodT富集,(miRNA),(mRNA+LncRNA+Pre-mRNA),真核转录组测序(人),AdvancedSummary,(200bp),(200bp),(200bp),.,原始测序数据,测序数据质量评估,参考序列比对分析,RNA-seq整体质量评估,mRNA分析,LncRNA分析,miRNA分析,mRNA-seq整体质量评估已知基因结构优化新基因预测反义转录本鉴定TSS和TTS位点统计可变剪切分析融合基因分析SNV和InDel分析,LncRNA-seq整体质量评估LncRNA序列拼接组装LncRNA位点及长度分析LncRNA分类LncRNA保守性分析,基因表达水平分析差异基因表达分析差异基因GO和KEGG分析蛋白互做网络分析,LncRNA表达水平分析LncRNA差异表达分析LncRNA靶