RNA-seq研究方法与策略-zzz.ppt

1、a,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,a,2,DNA makes RNA makes protein,mRNA是沟通DNA和蛋白质的“桥梁”,a,3,Messenger 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 express

2、ion. 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 target mRNAs to induce translat

3、ional repression, which is often linked to degradation. Long non-coding RNAs (long ncRNAs, lncRNA) are non-protein coding transcripts longer than 200 nucleotides.,Chris P. Ponting, Peter L. Oliver, and Wolf Reik. Evolution and Functions of Long Noncoding RNAs. Cell 136, 629641, February 20, 2009.,RN

4、A world is more colorful,a,4,Dual RNA-seq of pathogen and host. 10, 618630 (2012).,RNA Type,a,5,一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。,a,6,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、

5、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!,a,7,一个动物细胞中，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2 kb。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

6、约有11,000种，占到了mRNA数量的45%。,Challenge,a,8,Transcriptome The transcriptome is the complete set of transcripts in a cell, and their quantity, for a specific developmental stage or physiological condition. To catalogue all species of transcript (mRNAs, ncRNAs); To determine the transcriptional structure o

7、f genes (their start sites, 5 and 3 ends, splicing patterns and other post transcriptional modifications); To quantify the changing expression levels of each transcript during development and under different conditions.,RNA sequencing RNA-seq (RNA Sequencing), also called “Whole Transcriptome Shotgu

8、n Sequencing” (“WTSS”), is a technology that utilizes the capabilities of next generation sequencing to reveal a snapshot of RNA presence and quantity from a genome at a given moment in time.,a,9,但通常我们使用的是RNA-seq的狭义概念，亦即mRNA-seq。,Necsulea A, Kaessmann H. Evolutionary dynamics of coding and non-codin

9、g transcriptomes. Nat Rev Genet. 2014 Nov;15(11):734-48.,a,10,Trends in “Transcriptome and RNA-seq”,a,11,a,12,GENReports: Market 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; Key mutation对mRNA转录本表达量的影响如剪接位点、motif等位置的突变;,Why we need RNA/RNA-seq studies?,First,Second,a,15,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

10、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-seq Conclusion,Third,a,16,a,17,2. 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,a,18,Figure 1 RNA-seq work flow. Schematic diagram of RNA-seq library

11、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 molecules, such as mRNA or small RNA. mRNA is fragmented into a uniform siz

12、e 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; this information is important for RNA-seq data analysis. Mapping program

13、s 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. 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 trans

14、cripts. 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 assay

15、s. 13, 802807 (2012).,?,a,19,We 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 Proton, Pacific Biosci

16、ences 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 platforms.,a,20,For intac

17、t RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples.,a,21,a,22,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。,a,23,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

18、重复。,a,24,RNA Preparation,Isolate 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: Selec

19、tion 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 fragmentation,RNA enrichment methods Poly(A)-RNA selection - by hybridization to oligo-dT beads - mature mRN

20、A highly enriched - 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

21、kit - all non-rRNA 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.,a,25,a,26,Examples of good and poor quality RNA preps are shown in Fig

22、ure 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;,A,B,a,27,Prepare Libraries,First-strand synthesis (Reverse transcriptases,) Using oligo-dT to prime of

23、f 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

24、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 Sequencing adapters Rega

25、rdless 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, ligation) Preparation o

26、f stranded libraries Validation and Quantification,a,28,Table 3.1 List of functional elements contained in sequencing adapters.,Commercial kits,a,29,Sequencing,Choosing a sequencing platform Sample preparation and submission,Furthermore, the facility needs to know: The sequence of the sequence-primi

27、ng site. The length of the read you desire. Whether you 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-pri

28、ming site (i.e. a barcode).,Some general issues that need to 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

29、samples are suspended in appropriate buffers.,a,30,测序长度,测序数据,a,31,Analysis,Stereotypical RNA-seq Analysis Pipeline Demultiplex, 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). Ma

30、p (align) sequencing reads to reference genome or transcriptome. 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 treatm

31、ents. Perform multivariate statistical analysis/visualization to assess transcriptome-wide differences among samples.,a,32,Total RNA,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),Advanced Summary,(200bp),(200bp),(200bp),a,33,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