全转录组LncRNA与CircRNA测序分析策略

© 2010-2016 Novel Bio Co.,Ltd全转录组测序引爆点——LncRNA/CircRNA创新研究策略及解决方案© 2010-2016 Novel Bio Co.,Ltd生命体中的暗物质1RNA-seq之“暗物质”解决方案2lncRNA与circRNA的分析方案3目录CONTENTS© 2010-2016 Novel Bio Co.,Ltd生命体中的暗物质1RNA-seq之“暗物质”解决方案2lncRNA与circRNA的分析方案3目录CONTENTS© 2010-2016 Novel Bio Co.,Ltd非编码RNA的分类1. HousekeepingncRNA• tRNA,rRNA,snoRNA,snRNA• Translation,ribosomemature,RNAsplicing…etc2. SmallregulatoryRNA• miRNA(21-24nt),piRNA(26-31nt),siRNA(20-25ntDS)• developmentanddisease3. LongnoncodingRNA(lncRNA)• ≥200nt• MostprevalentclassofncRNA• XIST,HOTAIR…• developmentanddisease(1)DeSaccoet.al.Int.JournalofMol.Sci.(2012),13.(2) Taftetal.,JPathol2010;220:126–139,Non-codingRNAs:regulatorsofdisease(3) BakerM.LongnoncodingRNAs:thesearchforfunction.Nat.Meth.2011;8:379–383.16,592个已注释的ncRNA的主要分类lincRNA,5089AntisenseRNA,3230snRNA,1944microRNA,1756snoRNA,1521miscRNA,1187uncategorizedprocessedtranscript,933other,932© 2010-2016 Novel Bio Co.,Ltd非编码RNA的分类1. HousekeepingncRNA• tRNA,rRNA,snoRNA,snRNA• Translation,ribosomemature,RNAsplicing…etc2. SmallregulatoryRNA• miRNA(21-24nt),piRNA(26-31nt),siRNA(20-25ntDS)• developmentanddisease3. LongnoncodingRNA(lncRNA)• ≥200nt• MostprevalentclassofncRNA• XIST,HOTAIR…• developmentanddisease(1)DeSaccoet.al.Int.JournalofMol.Sci.(2012),13.(2) Taftetal.,JPathol2010;220:126–139,Non-codingRNAs:regulatorsofdisease(3) BakerM.LongnoncodingRNAs:thesearchforfunction.Nat.Meth.2011;8:379–383.4. circRNA• IntroniccircRNA，ExoniccircRNA• miRNASponges/RBPSponges• Cancer+Neuroscience© 2010-2016 Novel Bio Co.,LtdlncRNA的表达特征与mRNA相比，lncRNA一般表达量较低，而组织特异性更强Derrienetal,2012,GenomeResearchMoranN.Cabili,GENES&DEVELOPMENT，2011© 2010-2016 Novel Bio Co.,Ltd核内长链非编码RNA的功能u A.充当增强子enhancer(eRNA)招募RNA聚合酶IIu B.招募染色质修饰复合物u C.影响转录因子的招募u D.影响染色体的空间构象u E.影响RNA的剪接molecular cell 2016cell stem cell 2014Science 2012naturecommunications2013NatStructMolBiol.2016© 2010-2016 Novel Bio Co.,Ltd胞质中的lncRNAs功能u F.影响mRNA的稳定性u G.影响mRNA的翻译速率u H.作为ceRNA竞争性结合microRNAu I.部分lncRNA具有的短ORF，可以翻译成小肽Cell 2011; Cancer Cell 2014NATUREREVIEWSGENETICS2014Elife.2014© 2010-2016 Novel Bio Co.,LtdcircRNA相对于lncRNA的特征共价环化的RNA环细胞中稳定性极佳并且半衰期较长已知的circRNA已有150000个线性的RNA序列细胞中稳定性不佳已知的lncRNA已有100000个甚至更多lncRNA的特征circRNA的特征多miRNA结合位点——miRNA海绵机制&蛋白结合机制生命体中的暗物质1RNA-seq之“暗物质”解决方案2lncRNA与circRNA的分析方案3目录CONTENTS关于我们：烈冰团队4© 2010-2016 Novel Bio Co.,Ltd如何设计一次lncRNA的研究课题设计与样本准备测序与数据分析后期功能验证研究目标：circRNA，lncRNA？Novel，Known？实验手段：PolyA（+），Ribofree？样本选择：生物学重复？采样位置？研究目标：我的生物学表型是什么？分析手段：我希望分析到什么程度？是否需要定制化的讨论和分析？深入分析：是否需要分析最新的分析内容，如circRNA？研究目标：我希望验证到什么程度？实验手段：是否要补充一部分高通量测序的后期验证？© 2010-2016 Novel Bio Co.,LtdncRNA测序与数据分析流程© 2010-2016 Novel Bio Co.,Ltd1.用ribozerokit去除rRNA建库转录组寻找• 可以获得更多的ncRNA序列，适合预测新lncRNA• 同时获得mRNA数据2.polyA+富集建库• 仅能获得polyA+的lncRNA• 同时获得mRNA数据• 寻找已知的lncRNA或者只是研究的一小部分lncRNA测序分析策略环状RNA又如何？© 2010-2016 Novel Bio Co.,LtdcircRNA测序与数据分析流程© 2010-2016 Novel Bio Co.,Ltd1.用ribozerokit去除rRNA建库转录组寻找2.RNaseR去除线性RNA序列circRNA测序分析策略© 2010-2016 Novel Bio Co.,Ltd历代发表文章的建库统计更多的环状RNA被检测更准确的背景与表达量的关系Iju Chen, WIREs RNA 2016, 6:563–579. doi: 10.1002/wrna.1294 生命体中的暗物质1RNA-seq之“暗物质”解决方案2lncRNA与circRNA的分析方案3目录CONTENTS关于我们：烈冰团队4© 2010-2016 Novel Bio Co.,Ltd研究策略测序质量控制序列比对预测新lncRNA预测新circRNA得到已知lncRNAncRNA对功能影响ncRNA作用机制推测circRNA/lncRNA功能预测得到已知circRNA新序列预测测序数据处理数据库功能推测实验验证© 2010-2016 Novel Bio Co.,LtdlncRNA数据分析研究——KnownBioinformaOcs 2016NONCODEPRIMARY DATABASES1GENCODEEncyclopædia of genes and gene variantsh[p://(Derrien et al., 2012)2LNCipediaA database for annotated human lncRNA transcript sequences and structures.h[p://(Volders et al., 2013)3lncRNAdblncRNAdb is a database providing comprehensive annotaOons of funcOonal long noncoding RNAs (lncRNAs)h[p://(Amaral et al., 2011)4lncRNAMapFuncOonal Long Noncoding RNAs Databaseh[p://lncrnamap.mbc.nctu.edu.tw/php/(Chan et al., 2014)5lncRNomelncRNome is a comprehensive searchable biologically oriented knowledgebase for long noncoding RNAs in Humans.h[p://genome.igib.res.in/lncRNome/(BharOya et al., 2013)6NONCODE 4.0NONCODE provides an integraOve annotaOon of long noncoding RNAs.h[p://noncode.bioinfo.org.cn(Xie et al., 2014)7The FuncOonal lncRNA Data- baserepository of mammalian long non-protein coding transcripts that have been experimentally shown to be both noncoding and funcOonalh[p://(Niazi and Valadkhan, 2012)8NREDA database of long noncoding RNA expressionh[p://nred.mahcklab.com/cgi- bin/ncrnadb.pl(Dinger et al., 2009)SECONDARY INFORMATION DATABASES9ChIPBaseDatabase for annotaOng and exploring the expression profiles and the transcrip- Oonal regulaOon of lncRNAs and other ncRNAs.h[p://deepbase.sysu.edu.cn/chipbase/(Yang et al., 2013)10DIANA- LncBaseExperimentally verified and computaOonally predicted microRNA targets on long noncoding RNAs.(Paraskevopoulou et al., 2013)11GeneCards v3Integrated database of human genes that provides comprehensive, updated, and user-friendly informaOon on all known and predicted human genes.(Safran et al., 2010)12Linc2G