RNA-seq研究方法與策略-zzz

1、.,RNA-seq研究方法與策略,市場部 張壯壯 上海天昊生物科技有限公司,.,DNA makes RNA makes protein,mRNA是溝通DNA和蛋白質(zhì)的“橋梁”,.,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 expression. A non-cod

2、ing 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 repressi

3、on, 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.,RNA world is mor

4、e colorful,.,Dual RNA-seq of pathogen and host. 10, 618630 (2012).,RNA Type,.,一個(gè)典型的快速生長的哺乳動(dòng)物細(xì)胞培養(yǎng)中,每個(gè)細(xì)胞大約含有10-30 pg的RNA，而一個(gè)完全分化的原代細(xì)胞中，RNA的量要少得多大約每個(gè)細(xì)胞中RNA的含量小于1 pg。細(xì)胞中的RNA分子主要是tRNA和rRNA。mRNA大約占細(xì)胞中RNA總量的1-5%，但是具體的量取決于細(xì)胞類型和細(xì)胞的生理狀態(tài)。,.,RNA的特點(diǎn),分子相對(duì)較小，通常是單鏈； 周期短，降解快； 通常有特殊結(jié)構(gòu) (mRNA、miRNA、tRNA和rRNA)； 通常有前體，需要

5、剪切和修飾 (mRNA、miRNA、tRNA和rRNA)；,mRNA的特點(diǎn),5端帽子結(jié)構(gòu)和3端Poly A尾巴 分子長度一般介于500-10000nt 有前體，包含內(nèi)含子 能翻譯成功能蛋白,原核生物mRNA缺少cap和Poly-A tail的結(jié)構(gòu)!,.,一個(gè)動(dòng)物細(xì)胞中，大約有360,000個(gè)mRNA分子，組成了大約12,000個(gè)轉(zhuǎn)錄本，一個(gè)典型轉(zhuǎn)錄本的長度大約為2 kb。一些mRNA分子占到了總mRNA的3%，而其它的mRNA分子的含量低于0.01%。這些“稀有的”或者“低豐度”的mRNA分子在每個(gè)細(xì)胞中只有5-15個(gè)拷貝。但是，這些稀有的mRNA大約有11,000種，占到了mRNA數(shù)量的45

6、%。,Challenge,.,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 of genes (their start sit

7、es, 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 Shotgun Sequencing” (“WTSS”),

8、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.,.,但通常我們使用的是RNA-seq的狹義概念，亦即mRNA-seq。,Necsulea A, Kaessmann H. Evolutionary dynamics of coding and non-coding transcriptomes. Nat Rev

9、Genet. 2014 Nov;15(11):734-48.,.,Trends in “Transcriptome and RNA-seq”,.,.,GENReports: Market 某些特殊分子特征只能在RNA水平才能觀察到可變剪接、基因融合、RNA編輯等; Key mutation對(duì)mRNA轉(zhuǎn)錄本表達(dá)量的影響如剪接位點(diǎn)、motif等位置的突變;,Why we need RNA/RNA-seq studies?,First,Second,.,直接得到核酸序列信息，除了得到基因表達(dá)量的差異，更可以檢測(cè)RNA的結(jié)構(gòu)和結(jié)構(gòu)變異。 開放性的轉(zhuǎn)錄組分析：無需參考基因組信息，無需設(shè)計(jì)探針，不但能檢測(cè)

10、已知基因還能夠發(fā)現(xiàn)新的轉(zhuǎn)錄本。 在測(cè)序覆蓋率足夠大時(shí)能夠檢測(cè)到細(xì)胞中的低豐度轉(zhuǎn)錄本。 隨著測(cè)序深度的增加可以獲得更廣的動(dòng)態(tài)檢測(cè)范圍，能夠同時(shí)鑒定和定量高豐度轉(zhuǎn)錄本和低豐度轉(zhuǎn)錄本。 價(jià)格相對(duì)便宜,RNA-seq Conclusion,Third,.,.,2. RNA的提取與質(zhì)檢,3. 測(cè)序文庫的構(gòu)建,4. 上機(jī)測(cè)序與數(shù)據(jù)質(zhì)控,5. 數(shù)據(jù)分析與結(jié)果展示,1. 試驗(yàn)方案設(shè)計(jì),.,Figure 1 RNA-seq work flow. Schematic diagram of RNA-seq library construction. Total RNA is extracted from 300,0

11、00 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 distribution and the fragment size can be mon

12、itored 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 align reads to the reference genome and map s

13、plice 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 transcripts. The sequences of the reads can be mined

14、 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. 13, 802807 (2012).,?,.,We carried out replic

15、ate 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 Biosciences RS and Roche 454). The results show high int

16、raplatform (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.,.,For intact RNA, gene expression profiles from rRNA-depletion a

17、nd poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples.,.,.,重復(fù)的設(shè)置：技術(shù)重復(fù)、生物學(xué)重復(fù),技術(shù)誤差和個(gè)體差異可以通過設(shè)置重復(fù)進(jìn)行評(píng)估，但不能消除。 只有準(zhǔn)確平衡了技術(shù)誤差和個(gè)體差異，才能用RNA-seq結(jié)果解釋組間差異。,.,RNA-seq文庫構(gòu)建和測(cè)序的技術(shù)重復(fù)性皆為0.99以上，可以不設(shè)技術(shù)重復(fù)。,.,RNA Preparation,Isolate and purify RNA Solubilization Me

18、chanical 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: Selection of target RNAs via hybridization. Removal of non-target RNAs

19、 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 mRNA highly enriched- efficient for quantitation of gene expression le

20、vel- 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 retained: pre-mature mRNA, long non-coding RNA- neces

21、sary for prokaryotic organisms Small RNA extraction:- specific kits required to retain small RNA: Ambion mirVana kit - optional fine size-selection by gel.,.,.,Examples of good and poor quality RNA preps are shown in Figure A (agarose gel) and Figure B (Bioanalyzer trace).,RNA的操作本就是項(xiàng)復(fù)雜、精細(xì)的工作！,RNA質(zhì)量要

22、求：,Total RNA，溶解在H2O或TE (pH 8.0) 中; OD 260/280值應(yīng)在1.82.2 之間，RNA 28S:18S1.5，推薦 RIN7；無DNA污染; 最低濃度不低于100ng/L; 每個(gè)樣品總量不少于5g;,A,B,.,Prepare Libraries,First-strand synthesis (Reverse transcriptases,) Using oligo-dT to prime off of the poly-A tail of mature mRNA. Using random primers to prime at random positi

23、ons 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 adapter pre-ligated to the 5-end of the RNA template. Using a primer containing

24、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 Regardless of the platform, two types of sequence elements are required: (1) Terminal p

25、latform-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 libraries Validation and Quantification,.,Table 3.1 List of functional e

26、lements contained in sequencing adapters.,Commercial kits,.,Sequencing,Choosing a sequencing platform Sample preparation and submission,Furthermore, the facility needs to know: The sequence of the sequence-priming site. The length of the read you desire. Whether you want single-end or paired-end rea

27、ds. 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 to be considered are: That the

28、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.,.,測(cè)序長度,測(cè)序數(shù)據(jù),.,Analysis,Stereotypical RNA-se

29、q 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). Map (align) sequencing reads to reference genome or transcriptome. Annotate transcripts assembled

30、 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 assess transcriptome-wide diff

31、erences among samples.,.,Total RNA,oligodT磁珠富集mRNA,打斷、雙鏈cDNA合成,末端修復(fù)、加A加接頭,片段選擇,PCR擴(kuò)增、純化,rRNA 去除,文庫質(zhì)量檢測(cè),Illumina測(cè)序,片段大小篩選,oligodT富集,(miRNA),(mRNA+LncRNA+Pre-mRNA),真核轉(zhuǎn)錄組測(cè)序 (人),Advanced Summary,(200bp),(200bp),(200bp),.,原始測(cè)序數(shù)據(jù),測(cè)序數(shù)據(jù)質(zhì)量評(píng)估,參考序列比對(duì)分析,RNA-seq整體質(zhì)量評(píng)估,mRNA分析,LncRNA分析,miRNA分析,mRNA-seq整體質(zhì)量評(píng)估 已知基因結(jié)構(gòu)優(yōu)化 新基因預(yù)測(cè) 反義轉(zhuǎn)錄本鑒定 TSS和TTS位點(diǎn)統(tǒng)計(jì) 可變剪切分析 融合基因分析 SNV和InDel分析,LncRNA-seq整體質(zhì)量評(píng)估 LncRNA序列拼接組裝 LncRNA位點(diǎn)及長度分析 LncRNA分類 LncRNA保守性分析,基因表達(dá)水平分析