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1、RNA-seq研究方法与策略市场部张壮壮上海天昊生物科技有限公司DNAmakesRNAmakesproteinmRNA是沟通DNA和蛋白质的“桥梁”Messenger RNA (mRNA) isalargefamilyofRNAmoleculesthatconveygeneticinformationfromDNAtotheribosome,wheretheyspecifytheaminoacidsequenceoftheproteinproductsofgeneexpression.A non-coding RNA (ncRNA) is a functionalRNA molecule th
2、at is not translated into aprotein.microRNAs (miRNAs)Smallnon-codingRNAsof22nucleotidesthatareintegralcomponentsofRNA-inducedsilencingcomplex(RISC)andthatrecognizepartiallycomplementarytargetmRNAstoinducetranslationalrepression,whichisoftenlinkedtodegradation.Long non-coding RNAs(longncRNAs,lncRNA)a
3、renon-proteincodingtranscriptslongerthan200nucleotides.mRNACodingRNArRNAtRNAsnoRNAscaRNAsnRNANon-codingRNAirasiRNApiRNAsiRNAmiRNAstRNAanti-senselncRNAcircRNAChris P. Ponting, Peter L. Oliver, and Wolf Reik. Evolution and Functions of Long Noncoding RNAs. Cell 136, 629641, February 20, 2009.RNA world
4、 is more colorfulDualRNA-seqofpathogenandhost.10,618630(2012).RNAType一个典型人一个典型人类细胞的胞的RNA含量含量参数量每个细胞中的总RNA130pg细胞核中总RNA的比例14%细胞核中DNA:RNA2:1mRNA分子2x105-1x106mRNA常规大小1900nt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
5、取决于细胞类型和细胞的生理状态。RNA的特点l分子相对较小，通常是单链；l周期短，降解快；l通常有特殊结构(mRNA、miRNA、tRNA和rRNA)；l通常有前体，需要剪切和修饰(mRNA、miRNA、tRNA和rRNA)；mRNA的特点u5端帽子结构和3端PolyA尾巴u分子长度一般介于500-10000ntu有前体，包含内含子u能翻译成功能蛋白原核生物mRNA缺少cap和Poly-Atail的结构!基于丰度的mRNA分类丰度拷贝/细胞每个细胞中不同mRNA的数量每种mRNA的丰度低51511,0000.004%中等2004005000.1%高12,000200nt)Readlength5
6、0SE90PE50SE90PEIdentifynoveltranscriptsProfilingGenestructureSNP/SNVbiomarkerGenefusionRNA-seq TypeAlternative CommentmRNA-seq/LncRNA-seqpoly-A+mRNAandLncRNASmallRNA-seq(miRNA-seq)poly-A-miRNA,piRNA,.rmRNA-seqrRNA-codingandnon-codingRNAsTotalRNA-seqBothallRNAs,butmostofthemarerRNAsandtRNAs2.RNA的提取与质
7、检3. 测序文序文库的的构建构建4.上机测序与数据质控5.数据分析与结果展示1.试验方案设计普通普通转录组文文库LncRNA文文库Small RNA文文库Total RNAmRNANon-codingRNAmRNA文文库LncRNA-seqmiRNA-seqmRNA-seq真核真核链特异性文特异性文库真核原核De novo Assembly Transcript Re-sequencingFigure1RNA-seqworkflow.(a)Schematic diagram of RNA-seq library construction.Total RNA isextractedfrom300
8、,000cellsto3millioncells,andasmallaliquotisusedtomeasuretheintegrityoftheRNA.rRNAisthendepletedthroughoneofseveralmethodstoenrichsubpopulationofRNAmolecules,suchas mRNA or small RNA. mRNA is fragmented into auniform size distribution and the fragment size can bemonitored by RNA gel electrophoresis o
9、r AgilentBioanalyzer. ThecDNA isthenbuiltintoalibrary. ThesizedistributionpatternofthelibrarycanbecheckedbyAgilent Bioanalyzer; this information is important forRNA-seqdataanalysis.(b)Mappingprogramsalignreadstothereference genomeand map splice junctions. Gene expression can bequantifiedasabsolutere
10、adcountsornormalizedvaluessuchasRPKM.(c)IfRNA-seqdatasetsaredeepenoughandthereadsarelongenoughtomapenoughsplicejunctions,themappedreadscanbeassembledintotranscripts.(d)Thesequencesofthereadscanbeminedbycomparingthe transcriptome reads with the reference genome toidentify nucleotide variants that are
11、 either genomicvariants (for example, SNPs) or candidates for RNAediting.RNA-seq Workflow Technicalconsiderationsforfunctionalsequencingassays.13,802807(2012).?Wecarriedoutreplicateexperimentsacross15 laboratory sites using reference RNA standards totestfourprotocols(poly-A-selected,ribodepleted,siz
12、e-selectedanddegraded)onfivesequencingplatforms(IlluminaHiSeq,LifeTechnologiesPGMandProton,PacificBiosciencesRSandRoche454).Theresultsshowhigh intraplatform (Spearman rank R 0.86) and inter-platform (R 0.83) concordance for expression measures across the deep-count platforms, but highly variable eff
13、iciency and cost for splice junction and variant detection between all platforms.For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment aresimilar.Inaddition,rRNAdepletionenableseffectiveanalysisofdegradedRNAsamples.读长 (结构正确性) (表达量准确性) 通量Roche454读长很长(700bp)通量低(700M)测试费用很高
14、MiSeq读长中等(2300bp)通量中等(15G)测试费用中等HiSeq读长中等(2150bp)通量高(1.8T)测试费用低p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结果果变异异 组间差异+技术误差+个体误差实验目的源于技术源于不同个体技术重复评估生物学重复评估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。RNAPreparationIsolate and purify RNASolubilizationMechanicalhom
15、ogenizationRecoveryofRNAfromlysate:Organicextraction/Solid-phaseextractionQuantitationandQualityAssessmentTarget enrichment：ThefourmethodsthatarecommonlyusedtoenrichspecificclassesofRNAsare:SelectionoftargetRNAsviahybridization.Removalofnon-targetRNAsviahybridization.Copy-numbernormalizationviaduple
16、x-specificnucleasedigestion.Targetenrichmentviasize-selectionRNA fragmentationRNA enrichment methods lPoly(A)-RNAselection-byhybridizationtooligo-dTbeads-maturemRNAhighlyenriched-efficientforquantitationofgeneexpressionlevel-limitation:3biascorrelatingwithRNAdegradationlrRNAdepletion:-byhybridizatio
17、ntobead-boundrRNAprobes-rRNAsequence-dependentandspecies-specific-commercialkits:InvitrogenRibo-minuskit;EpicenterRibo-Zerokit-allnon-rRNAretained:pre-maturemRNA,longnon-codingRNA- necessary for prokaryotic organisms lSmallRNAextraction:-specifickitsrequiredtoretainsmallRNA:AmbionmirVanakit-optional
18、finesize-selectionbygel.ExamplesofgoodandpoorqualityRNAprepsareshownin Figure A (agarose gel) and Figure B (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;ABPrepareLibrariesFirst-strandsynthesis(Reve
19、rsetranscriptases,)Usingoligo-dTtoprimeoffofthepoly-AtailofmaturemRNA.UsingrandomprimerstoprimeatrandompositionsalongtheRNAmolecule.PrimingoffofoligosthatareligatedontotheendsoftheRNA.Second-strandsynthesis(DNApolymerase)SynthesisbyRNAnickinganddisplacement.Using an oligo that is complementary to an
20、 adapter pre-ligated to the 5-end of the RNAtemplate.Usingaprimercontaininga3-oligo-dG(thismethod,referredtoasSMART)takesadvantageofthephenomenonthattheMMLVreverse-transcriptaseleavesaterminalnon-templatepoly-dC3-overhang).FragmentationofcDNASequencingadaptersRegardlessoftheplatform,twotypesofsequen
21、ceelementsarerequired:(1)Terminalplatform-dependentsequencesthatarerequiredforclonalamplificationandattachmenttothesequencingsupport.(2)Sequencesforprimingthesequencingreaction.Additionofadapters(RT/PCR,ligation)PreparationofstrandedlibrariesValidation and QuantificationAdapter elementRequirementLoc
22、ationFunctionAmplification elementRequired5and3terminusClonalamplificationoftheconstructPrimary sequencing priming siteRequiredAdjacenttotheinsertInitiatingtheprimarysequencingreactionBarcode/IndexOptional5-endoftheinsert/BetweenthesequencingprimingsiteanditsrespectiveamplificationelementProvidesaun
23、iquelabeltosequencesfromdifferentsamples.Allowspoolingofmultipleexperimentsinasinglesequencingreaction.Paired-end sequencing priming siteOptionalAdjacenttotheinsertonthesideoppositeoftheprimarysequencingprimingsiteSequencingintotheinsertontheendoppositeoftheprimaryreadIndex sequencing priming siteOp
24、tionalComplementarytothe5-endofthesequencingprimingsiteSequencingoftheindexTable 3.1 List of functional elements contained in sequencing adapters.CommercialkitsSequencingChoosingasequencingplatformSamplepreparationandsubmissionFurthermore,thefacilityneedstoknow:1.Thesequenceofthesequence-primingsite
25、.2.Thelengthofthereadyoudesire.3.Whetheryouwantsingle-endorpaired-endreads.4.Whetherthereisabarcodeorindexsequence.5.IfusingIlluminasequencingthefacilityalsoneedstobenotifiediftheinsertscontainaregionoflowsequencecomplexityimmediatelyafterthesequence-primingsite(i.e.abarcode).Somegeneralissuesthatne
26、edtobeconsideredare:1.Thatthesamplesarecleanandfreeofmajorcontaminants.2.TheprimaryDNAmoleculescontaininsertsofthecorrectsize.3.TheprimaryDNAmoleculeshaveadaptersoneachend.4.Thesampleconcentrationisappropriate.5.Thesamplesaresuspendedinappropriatebuffers.测序长度读长特点特点主要主要应用用150bp读长较长测序深度较高基因表达检测175bp21
27、00bp综合型基因表达检测基因结构鉴定2125bp2150bp2300bp读长较长测序深度较低序列重头拼接转录组组装测序数据基因数目基因数目研究目的及相研究目的及相应测序深度序深度基因表达定量基因结构研究细菌1,500-4,0002-4Mreads1-2Gbdata真菌5,000-13,0006-10Mreads2-4Gbdata高等植物20,000-35,00010-20Mreads5-10Gbdata高等动物30,00010-20Mreads5-10GbdataAnalysisStereotypical RNA-seq Analysis Pipeline1.Demultiplex,filt
28、er,andtrimsequencingreads.2.Normalizesequencingreads(ifperformingde novoassembly).3.de novoassemblyoftranscripts(ifareferencegenomeisnotavailable).4.Map(align)sequencingreadstoreferencegenomeortranscriptome.5.Annotatetranscriptsassembledortowhichreadshavebeenmapped.6.Countmappedreadstoestimatetransc
29、riptabundance.7.Performstatistical analysistoidentify differential expression(ordifferential splicing)amongsamplesortreatments.8.Perform multivariate statistical analysis/visualization to assess transcriptome-widedifferencesamongsamples.Total RNAoligodT磁珠富集磁珠富集mRNA打断、双打断、双链cDNA合成合成末端修复、加末端修复、加A加接加接头
30、片段片段选择PCR扩增、增、纯化化rRNA 去除去除文文库质量量检测Illumina测序序片段大小片段大小筛选oligodT富集不不带polyA的的RNA(LncRNA)带polyA的的RNA(Poly A (mRNA+LncRNA)(miRNA)带polyA的的RNA(PolyA (mRNA+LncRNA)(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lmRNA-seq整体质量评估l已知基因
31、结构优化l新基因预测l反义转录本鉴定lTSS和TTS位点统计l可变剪切分析l融合基因分析lSNV和InDel分析lLncRNA-seq整体质量评估lLncRNA序列拼接组装lLncRNA位点及长度分析lLncRNA分类lLncRNA保守性分析l基因表达水平分析l差异基因表达分析l差异基因GO和KEGG分析l蛋白互做网络分析lLncRNA表达水平分析lLncRNA差异表达分析lLncRNA靶基因预测lLncRNA靶基因功能分析lLncRNA与靶基因调控网络分析lmiRNA表达水平分析lmiRNA差异表达分析lmiRNA靶基因预测lmiRNA靶基因功能分析lmiRNA与靶基因调控网络分析lmiRN
32、A-seq整体质量评估l已知miRNA鉴定l新miRNA预测lSmallRNA统计lmiRNA家族分析lmiRNA结果可视化特异性研究：设计的样本、准确的数据CaseStudy特征性研究：大样本、大数据April20102012Octorber2011Octorber2011September2012March20092012August2014December2014January2014January2015May2013April2014n更严谨的实验设计(生物学重复、对照、大样本量)及与之匹配的分析策略；n与其它组学整合分析(miRNA-seq、LncRNA-seq、BS-seq、re
33、sequencinge.g.)；n单细胞RNA-seqn后续功能研究；现阶段，RNA-seq已是一种常规的实验手段，不再是一种实验目的。研究目的决定研究目的决定实验策略策略1.RNA分子的获得：mRNA、LncRNA、miRNA2.文库的类型：普通文库、链特异性文库3.测序类型：读长、单端/双端、深度4.分析内容：表达、新转录本、可变剪切、融合基因、变异分析实验策略多组学联合分析-全转录组分析l基因表达注释l差异表达基因分析l差异基因表达模式分析lGO和Pathway富集分析l鉴定LncRNAlLncRNA定量及差异表达分析lLncRNA靶基因预测l靶基因GO和Pathway分析lCoding
34、-noncoding共表达注释l基于互补序列的LncRNA-mRNA互作分析lccRNA分析l差异miRNA与差异靶基因表达相关性分析l差异miRNA与差异基因表达网络图谱分析l差异miRNA靶基因GO与差异基因GO注释的关联分析l差异miRNA靶基因Pathway与差异基因Pathway注释的关联分析lmiRNA表达谱构建l新miRNA预测lmiRNA靶基因预测l靶基因GO和KEGG注释lmiRNA差异分析与聚类分析miRNA-seqmRNA-seqLncRNA-seqmiRNA与mRNA联合分析LncRNA与mRNA联合分析LncRNA与miRNA联合分析Thanks for your attention!
