RNA-seq研究方法与策略-zzz

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12021/8/14DNA makes RNA makes proteinmRNA是沟通DNA和蛋白质的“桥梁”22021/8/14Messenger 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 protein products of gene expression.A

2、 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 target mRNAs to induce translational re

3、pression, which is often linked to degradation.Long non-coding RNAs (long ncRNAs, lncRNA) are non-protein coding transcripts longer than 200 nucleotides. mRNACodingRNArRNAtRNAsnoRNAscaRNAsnRNANon-codingRNAirasiRNApiRNAsiRNAmiRNAstRNAanti-senselncRNAcircRNARNA world is more colorful32021/8/14Dual RNA

4、-seq of pathogen and host. 10, 618630 (2012).RNA Type42021/8/14一个典型人类细胞的一个典型人类细胞的RNA含量含量参数量每个细胞中的总RNA130 pg细胞核中总RNA的比例14%细胞核中DNA:RNA 2:1mRNA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%

5、，但是具体的量取决于细胞类型和细胞的生理状态。52021/8/14RNA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u 5端帽子结构和3端Poly A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-A tail的结构! 62021/8/14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

6、%中等2004005000.1%高12,000 200nt)Read length50SE90PE50SE90PEIdentify novel transcriptsProfilingGene 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 Tota

7、l RNA-seqBothall RNAs, but most of them are rRNAs and tRNAs 162021/8/142. RNA的提取与质检3. 测序文库的测序文库的构建构建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库转录组文库LncRNA文库文库Small RNA文库文库Total RNAmRNANon-coding RNAmRNA文库文库LncRNA-seqmiRNA-seqmRNA-seq真核链特异性文库真核链特异性文库真核原核De novo Assembly Transcript Re-sequencing172021/

8、8/14Figure 1 RNA-seq work flow. (a) Schematic diagram of RNA-seq library construction. 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 mo

9、lecules, such as mRNA or small RNA. mRNA is fragmented into a uniform size distribution 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;

10、 this information is important for RNA-seq data analysis. (b)Mapping programs align reads to the reference genome and map splice junctions. Gene expression can be quantified as absolute read counts or normalized values such as RPKM. (c) If RNA-seq data sets are deep enough and the reads are long eno

11、ugh to map enough splice junctions, the mapped reads can be assembled into transcripts. (d)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

12、 editing.RNA-seq Workflow Technical considerations for functional sequencing assays. 13, 802807 (2012).?182021/8/14We 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

13、sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, 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 an

14、d cost for splice junction and variant detection between all platforms.192021/8/14For 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.202021/8/14读长 (结构正确性) (表达量准确性) 通量Roche 454读

15、长很长 (700bp)通量低 (700M)测试费用很高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212021/8/14p 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222021/8/14RNA-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。 2

16、32021/8/14RNA PreparationIsolate and purify RNA Solubilization Mechanical homogenization Recovery of RNA from lysate: Organic extraction/Solid-phase extraction Quantitation and Quality Assessment Target enrichment：The four methods that are commonly used to enrich specific classes of RNAs are:Selecti

17、on of target RNAs via hybridization.Removal of non-target RNAs via hybridization.Copy-number normalization via duplex-specific nuclease digestion.Target enrichment via size-selection RNA fragmentationRNA enrichment methods lPoly(A)-RNA selection- by hybridization to oligo-dT beads- mature mRNA highl

18、y enriched- efficient for quantitation of gene expression level- limitation: 3 bias correlating with RNA degradation lrRNA 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 n

19、on-rRNA retained: pre-mature mRNA, long non-coding RNA- necessary for prokaryotic organisms lSmall RNA extraction:- specific kits required to retain small RNA: Ambion mirVana kit - optional fine size-selection by gel.242021/8/14252021/8/14Examples of good and poor quality RNA preps are shown in Figu

20、re 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;AB262021/8/14Prepare LibrariesFirst-strand synthesis (Reverse transcriptases,)Using oligo-dT to prime off

21、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 oligo that is complementary to an adap

22、ter 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 cDNASequencing adaptersRegardless of t

23、he 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, ligation)Preparation of stranded li

24、brariesValidation and Quantification272021/8/14Adapter elementRequirementLocationFunctionAmplification elementRequired5 and 3 terminusClonal amplification of the constructPrimary sequencing priming siteRequiredAdjacent to the insertInitiating the primary sequencing reactionBarcode/IndexOptional5-end

25、 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 priming siteOptionalAdjacent to the insert on the side

26、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 functional elements contained in sequencing adapters

27、.Commercial kits282021/8/14Sequencing Choosing a sequencing platform Sample preparation and submissionFurthermore, the facility needs to know:1. The sequence of the sequence-priming site.2. The length of the read you desire.3. Whether you want single-end or paired-end reads.4. Whether there is a bar

28、code or index sequence.5. 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 to be considered are:1.That the samples are clean and fr

29、ee of major contaminants.2.The primary DNA molecules contain inserts of the correct size.3.The primary DNA molecules have adapters on each end.4.The sample concentration is appropriate.5.The samples are suspended in appropriate buffers.292021/8/14测序长度读长读长特点特点主要应用主要应用150bp读长较长测序深度较高基因表达检测175bp2100bp综

30、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应测序深度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 data302021/8/14AnalysisStereotypical RNA-seq Analysis Pipeline1.

31、 Demultiplex, filter, and trim sequencing reads.2. Normalize sequencing reads (if performing de novo assembly).3. de novo assembly of transcripts (if a reference genome is not available).4. Map (align) sequencing reads to reference genome or transcriptome.5. Annotate transcripts assembled or to whic

32、h reads have been mapped.6. Count mapped reads to estimate transcript abundance.7. Perform statistical analysis to identify differential expression (or differential splicing) among samples or treatments.8. Perform multivariate statistical analysis/visualization to assess transcriptome-wide differenc

33、es among samples.312021/8/14Total RNAoligodT磁珠富集磁珠富集mRNA打断、双链打断、双链cDNA合成合成末端修复、加末端修复、加A加接头加接头片段选择片段选择PCR扩增、纯化扩增、纯化rRNA 去除去除文库质量检测文库质量检测Illumina测序测序片段大小筛选片段大小筛选oligodT富集不带不带polyA的的RNA(LncRNA)带带polyA的的RNA(Poly A (mRNA+LncRNA)(miRNA)带带polyA的的RNA(PolyA (mRNA+LncRNA)(mRNA+LncRNA+Pre-mRNA) 真核转录组测序 (人)Adva

34、nced Summary(200bp)(200bp)(200bp)322021/8/14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l mRNA-seq整体质量评估l 已知基因结构优化l 新基因预测l 反义转录本鉴定l TSS和TTS位点统计l 可变剪切分析l 融合基因分析l SNV和InDel分析l LncRNA-seq整体质量评估l LncRNA序列拼接组装l LncRNA位点及长度分析l LncRNA分类l LncRNA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l LncRNA表达水平分析l LncRNA差异表达分析l LncRNA靶基因预测l LncRNA靶基因功能分析l LncRNA与靶基因调控网络分析l miRNA表达水平分析l miRNA差异表达分析l miRNA