RNAseq研究方法與策略zzz課件

1、RNA-seq研究方法與策略市場部 張壯壯zhangzz上海天昊生物科技有限公司RNA-seq研究方法與策略市場部 張壯壯DNA makes RNA makes proteinmRNA是溝通DNA和蛋白質(zhì)的“橋梁”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一個(gè)典型人類細(xì)胞的RNA含量參數(shù)量每個(gè)細(xì)胞中的總RNA

5、130 pg細(xì)胞核中總RNA的比例14%細(xì)胞核中DNA:RNA2:1mRNA分子2 x 105- 1 x 106mRNA常規(guī)大小1900 nt一個(gè)典型的快速生長的哺乳動物細(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)。一個(gè)典型人類細(xì)胞的RNA含量參數(shù)量每個(gè)細(xì)胞中的總RNA1RNA的特點(diǎn)分子相對較小，通常是單鏈；周期短，降解快；通常有特殊結(jié)構(gòu) (mRNA、miRNA、tRNA和

6、rRNA)；通常有前體，需要剪切和修飾 (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)! RNA的特點(diǎn)分子相對較小，通常是單鏈；mRNA的特點(diǎn)5端帽基于豐度的mRNA分類豐度拷貝/細(xì)胞每個(gè)細(xì)胞中不同mRNA的數(shù)量每種mRNA的豐度低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的提取與質(zhì)檢3. 測序文庫的構(gòu)建4. 上機(jī)測序與數(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)普通轉(zhuǎn)錄組文庫LncRNA文庫Small RNA文庫Total RNAmRNANon-coding RNAmRNA文庫LncRNA-seqmiRNA-seqmRNA-seq真核鏈特異性文庫真核原核De novo Assembly Transcript Re-sequencing2. RNA的提取與質(zhì)檢3. 測序文庫的構(gòu)建4. 上機(jī)測序與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讀長 (結(jié)構(gòu)正確性) (表達(dá)量準(zhǔn)確性) 通量Roche 454讀長很長 (700bp)通量低 (700M)測試費(fèi)用很高M(jìn)i

16、Seq讀長中等 (2300bp)通量中等 (15G)測試費(fèi)用中等HiSeq讀長中等 (2150bp)通量高(1.8T)測試費(fèi)用低讀長 (結(jié)構(gòu)正確性) 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結(jié)果變異 組間差異 + 技術(shù)誤差 + 個(gè)體誤差實(shí)驗(yàn)?zāi)康脑从诩夹g(shù)源于不同個(gè)體技術(shù)重復(fù)評估生物學(xué)重復(fù)評估重復(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)RNA-seq文庫構(gòu)建和測序的技術(shù)重復(fù)性皆為0.99以上，可以不設(shè)技術(shù)重復(fù)。 RNA-seq文庫構(gòu)建和測序的技術(shù)重復(fù)

17、性皆為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研究方法與策略-zzzExamples of good and poor quality RNA preps

21、 are shown in Figure A (agarose gel) and Figure B (Bioanalyzer trace).RNA的操作本就是項(xiàng)復(fù)雜、精細(xì)的工作！RNA質(zhì)量要求：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;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測序長度讀長特點(diǎn)主要應(yīng)用150bp讀長較長測序深度較高基因表達(dá)檢測175bp2100bp綜合型基因表達(dá)檢測基因結(jié)構(gòu)鑒定2125bp2150bp2300bp讀長較長測序深度較低序列重頭拼接轉(zhuǎn)錄組組裝測序數(shù)據(jù)基因數(shù)目研究目的及相應(yīng)測序深度基因表達(dá)定量基因結(jié)構(gòu)研究細(xì)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高等動物30,00010-20M reads5-10Gb data測序長度讀長特點(diǎn)主要應(yīng)用150bp讀長

32、較長基因表達(dá)檢測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Illumina測序片段大小篩選oligodT富集不帶polyA的RNA(LncRNA)帶polyA的RNA(Poly A (mRNA+LncRNA)(miRNA)帶polyA的RNA(PolyA (mRNA+LncRNA)(mRNA+LncRNA+Pre-mRNA) 真核轉(zhuǎn)錄組測序 (人)Adv

35、anced Summary(200bp)(200bp)(200bp)Total RNAoligodT磁珠富集mRNA打斷、雙鏈c原始測序數(shù)據(jù)測序數(shù)據(jù)質(zhì)量評估參考序列比對分析RNA-seq整體質(zhì)量評估m(xù)RNA分析LncRNA分析miRNA分析mRNA-seq整體質(zhì)量評估已知基因結(jié)構(gòu)優(yōu)化新基因預(yù)測反義轉(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)水平分析差異基因表達(dá)分析差異基因GO和KEGG分析蛋白互做網(wǎng)絡(luò)分析LncRNA表達(dá)水平分析LncRNA差異表達(dá)分析LncRNA靶基因預(yù)測LncRNA靶基因功能分析LncRNA