RNAseq研究方法与策略zzz课件

1、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RNA-seq研究方法与策略市场部 张壮壮DNA makes RNA makes proteinmRNA是沟通DNA和蛋白质的“桥梁”DNA makes RNA makes proteinmMessenger RNA (mRNA) is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the p

2、rotein products of gene expression.A non-coding RNA (ncRNA) is a functional RNA molecule that is not translated into a protein.microRNAs (miRNAs)Small non-coding RNAs of 22 nucleotides that are integral components of RNA-induced silencing complex (RISC) and that recognize partially complementary tar

3、get mRNAs to induce translational repression, which is often linked to degradation.Long non-coding RNAs (long ncRNAs, lncRNA) are non-protein coding transcripts longer than 200 nucleotides. mRNACodingRNArRNAtRNAsnoRNAscaRNAsnRNANon-codingRNAirasiRNApiRNAsiRNAmiRNAstRNAanti-senselncRNAcircRNAChris P.

4、 Ponting, Peter L. Oliver, and Wolf Reik. Evolution and Functions of Long Noncoding RNAs. Cell 136, 629641, February 20, 2009.RNA world is more colorfulMessenger RNA (mRNA) is a largDual RNA-seq of pathogen and host. 10, 618630 (2012).RNA TypeDual RNA-seq of pathogen and h一个典型人类细胞的RNA含量参数量每个细胞中的总RNA

5、130 pg细胞核中总RNA的比例14%细胞核中DNA:RNA2:1mRNA分子2 x 105- 1 x 106mRNA常规大小1900 nt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10-30 pg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1 pg。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。一个典型人类细胞的RNA含量参数量每个细胞中的总RNA1RNA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

6、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Poly A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-A tail的结构! RNA的特点分子相对较小，通常是单链；mRNA的特点5端帽基于丰度的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12,000 200nt)Read length50SE90PE50SE90PEIdentify novel transcript

7、sProfilingGene structure SNP/SNVbiomarkerGene fusionRNA-seq TypeAlternative CommentmRNA-seq/LncRNA-seqpoly-A+mRNA and LncRNASmall RNA-seq (miRNA-seq)poly-A- miRNA, piRNA, . rmRNA-seqrRNA-coding and non-coding RNAs Total RNA-seqBothall RNAs, but most of them are rRNAs and tRNAs ApplicationsRNA-SeqSma

8、ll RNALn2. 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. 试验方案设计普通转录组文库LncRNA文库Small RNA文库Total RNAmRNANon-coding RNAmRNA文库LncRNA-seqmiRNA-seqmRNA-seq真核链特异性文库真核原核De novo Assembly Transcript Re-sequencing2. RNA的提取与质检3. 测序文库的构建4. 上机测序与Figure 1 RNA-seq work flow. Schematic diagram of RNA-seq library con

9、struction. Total RNA is extracted from 300,000 cells to 3 million cells, and a small aliquot is used to measure the integrity of the RNA. rRNA is then depleted through one of several methods to enrich subpopulation of RNA molecules, such as mRNA or small RNA. mRNA is fragmented into a uniform size d

10、istribution and the fragment size can be monitored by RNA gel electrophoresis or Agilent Bioanalyzer. The cDNA is then built into a library. The size distribution pattern of the library can be checked by Agilent Bioanalyzer; this information is important for RNA-seq data analysis. Mapping programs a

11、lign reads to the reference genome and map splice junctions. Gene expression can be quantified as absolute read counts or normalized values such as RPKM. If RNA-seq data sets are deep enough and the reads are long enough to map enough splice junctions, the mapped reads can be assembled into transcri

12、pts. The sequences of the reads can be mined by comparing the transcriptome reads with the reference genome to identify nucleotide variants that are either genomic variants (for example, SNPs) or candidates for RNA editing.RNA-seq Workflow Technical considerations for functional sequencing assays. 1

13、3, 802807 (2012).?Figure 1 RNA-seq work flow. RNWe carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribodepleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Pro

14、ton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R 0.86) and inter-platform (R 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platfo

15、rms.We carried out replicate experFor intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples.For intact RNA, gene expressio读长 (结构正确性) (表达量准确性) 通量Roche 454读长很长 (700bp)通量低 (700M)测试费用很高Mi

16、Seq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)测试费用低读长 (结构正确性) 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结果变异 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。 RNA-seq文库构建和测序的技术重复

17、性皆为0.99以上，可RNA PreparationIsolate and purify RNASolubilizationMechanical homogenizationRecovery of RNA from lysate: Organic extraction/Solid-phase extractionQuantitation and Quality AssessmentTarget enrichment：The four methods that are commonly used to enrich specific classes of RNAs are:Selection o

18、f target RNAs via hybridization.Removal of non-target RNAs via hybridization.Copy-number normalization via duplex-specific nuclease digestion.Target enrichment via size-selectionRNA fragmentationRNA enrichment methods Poly(A)-RNA selection- by hybridization to oligo-dT beads- mature mRNA highly enri

19、ched- efficient for quantitation of gene expression level- limitation: 3 bias correlating with RNA degradation rRNA depletion:- by hybridization to bead-bound rRNA probes- rRNA sequence-dependent and species-specific- commercial kits: Invitrogen Ribo-minus kit; Epicenter Ribo-Zero kit - all non-rRNA

20、 retained: pre-mature mRNA, long non-coding RNA- necessary for prokaryotic organisms Small RNA extraction:- specific kits required to retain small RNA: Ambion mirVana kit - optional fine size-selection by gel.RNA PreparationIsolate and purRNA-seq研究方法与策略-zzzExamples of good and poor quality RNA preps

21、 are shown in Figure A (agarose gel) and Figure B (Bioanalyzer trace).RNA的操作本就是项复杂、精细的工作！RNA质量要求：Total RNA，溶解在H2O或TE (pH 8.0) 中; OD 260/280值应在1.82.2 之间，RNA 28S: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;ABExamples of good and poor qualPrepare LibrariesFirst-strand synthesis (Reverse transcript

22、ases,)Using oligo-dT to prime off of the poly-A tail of mature mRNA.Using random primers to prime at random positions along the RNA molecule.Priming off of oligos that are ligated onto the ends of the RNA.Second-strand synthesis (DNA polymerase)Synthesis by RNA nicking and displacement.Using an olig

23、o that is complementary to an adapter pre-ligated to the 5-end of the RNA template.Using a primer containing a 3-oligo-dG (this method, referred to as SMART) takes advantage of the phenomenon that the MMLV reverse-transcriptase leaves a terminal non-template poly-dC 3-overhang).Fragmentation of cDNA

24、Sequencing adaptersRegardless of the platform, two types of sequence elements are required: (1) Terminal platform-dependent sequences that are required for clonal amplification and attachment to the sequencing support. (2) Sequences for priming the sequencing reaction.Addition of adapters (RT/PCR, l

25、igation)Preparation of stranded librariesValidation and QuantificationPrepare LibrariesFirst-strand Adapter elementRequirementLocationFunctionAmplification elementRequired5 and 3 terminusClonal amplification of the constructPrimary sequencing priming siteRequiredAdjacent to the insertInitiating the

26、primary sequencing reactionBarcode/IndexOptional5-end of the insert/Between the sequencing priming site and its respective amplification elementProvides a unique label to sequences from different samples. Allows pooling of multiple experiments in a single sequencing reaction.Paired-end sequencing pr

27、iming siteOptionalAdjacent to the insert on the side opposite of the primary sequencing priming siteSequencing into the insert on the end opposite of the primary readIndex sequencing priming siteOptionalComplementary to the 5-end of the sequencing priming siteSequencing of the indexTable 3.1 List of

28、 functional elements contained in sequencing adapters.Commercial kitsAdapter elementRequirementLocaSequencingChoosing a sequencing platformSample preparation and submissionFurthermore, the facility needs to know:The sequence of the sequence-priming site.The length of the read you desire.Whether you

29、want single-end or paired-end reads.Whether there is a barcode or index sequence.If using Illumina sequencing the facility also needs to be notified if the inserts contain a region of low sequence complexity immediately after the sequence-priming site (i.e. a barcode).Some general issues that need t

30、o be considered are:That the samples are clean and free of major contaminants.The primary DNA molecules contain inserts of the correct size.The primary DNA molecules have adapters on each end.The sample concentration is appropriate.The samples are suspended in appropriate buffers.SequencingChoosing

31、a sequencin测序长度读长特点主要应用150bp读长较长测序深度较高基因表达检测175bp2100bp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研究目的及相应测序深度基因表达定量基因结构研究细菌1,500-4,0002-4M reads1-2Gb data真菌5,000-13,0006-10M reads2-4Gb data高等植物20,000-35,00010-20M reads5-10Gb data高等动物30,00010-20M reads5-10Gb data测序长度读长特点主要应用150bp读长

32、较长基因表达检测1AnalysisStereotypical RNA-seq Analysis PipelineDemultiplex, filter, and trim sequencing reads.Normalize sequencing reads (if performing de novo assembly).de novo assembly of transcripts (if a reference genome is not available).Map (align) sequencing reads to reference genome or transcriptom

33、e.Annotate transcripts assembled or to which reads have been mapped.Count mapped reads to estimate transcript abundance.Perform statistical analysis to identify differential expression (or differential splicing) among samples or treatments.Perform multivariate statistical analysis/visualization to a

34、ssess transcriptome-wide differences among samples.AnalysisStereotypical RNA-seq Total RNAoligodT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富集不带polyA的RNA(LncRNA)带polyA的RNA(Poly A (mRNA+LncRNA)(miRNA)带polyA的RNA(PolyA (mRNA+LncRNA)(mRNA+LncRNA+Pre-mRNA) 真核转录组测序 (人)Adv

35、anced Summary(200bp)(200bp)(200bp)Total RNAoligodT磁珠富集mRNA打断、双链c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靶基因预测LncRNA靶基因功能分析LncRNA