RNA-seq研究方法与策略-zzzppt课件

RNA seq研究方法与策略 市场部 DNAmakesRNAmakesprotein mRNA是沟通DNA和蛋白质的 桥梁 MessengerRNA mRNA isalargefamilyofRNAmoleculesthatconveygeneticinformationfromDNAtotheribosome wheretheyspecifytheaminoacidsequenceoftheproteinproductsofgeneexpression Anon codingRNA ncRNA isafunctionalRNAmoleculethatisnottranslatedintoaprotein microRNAs miRNAs Smallnon codingRNAsof 22nucleotidesthatareintegralcomponentsofRNA inducedsilencingcomplex RISC andthatrecognizepartiallycomplementarytargetmRNAstoinducetranslationalrepression whichisoftenlinkedtodegradation Longnon codingRNAs longncRNAs lncRNA arenon proteincodingtranscriptslongerthan200nucleotides ChrisP Ponting PeterL Oliver andWolfReik EvolutionandFunctionsofLongNoncodingRNAs Cell136 629 641 February20 2009 RNAworldismorecolorful DualRNA seqofpathogenandhost 10 618 630 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 5 and3 ends 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 TechAnalysis ProducedbyEnalRazvi Ph D 2014 ProvidesaPictureofResearchEffortsbyTypeofNucleicAcid Weasked WhatisyourmajorapplicationforRNA Seq 1 RNA在生命过程中具有重要地位 2 对DNA预测基因的修正 3 疾病 肿瘤研究中具有重要作用 4 生物与环境的相互作用 5 基因表达调控研究 基因组被认为是不变的 但是某种实验条件却能显著改变基因的表达量 如药物处理VS 正常样品 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 13 802 807 2012 Wecarriedoutreplicateexperimentsacross15laboratorysitesusingreferenceRNAstandardstotestfourprotocols poly A selected ribodepleted size selectedanddegraded onfivesequencingplatforms IlluminaHiSeq LifeTechnologiesPGMandProton PacificBiosciencesRSandRoche454 Theresultsshowhighintraplatform SpearmanrankR 0 86 andinter platform R 0 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 3 biascorrelatingwithRNAdegradationrRNAdepletion 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 8 2 2之间 RNA28S 18S 1 5 推荐RIN 7 无DNA污染 最低浓度不低于100ng L 每个样品总量不少于5 g A B PrepareLibraries First strandsynthesis Reversetranscriptases Usingoligo dTtoprimeoffofthepoly AtailofmaturemRNA Usingrandomprimerstoprimeat random positionsalongtheRNAmolecule 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靶基