复旦大学RNA-seq 转录组测序培训班.pptx

理解复杂的转录组：从头到尾;提 纲;RNA-seq背景介绍;基因和环境共同作用决定表型;DNA;;下一代测序(二代/三代测序、深度测序)技术为研究基因型和表型的关系提供了有力手段;下一代测序技术作为研究手段的优点;454 FLX;Mortazavi et al. Nature Methods, 2008;RNA-seq的优点;RNA-seq实验方法简介;目的不同决定了RNA-seq文库构建流程不同;总RNA的纯化处理是检测polyA+ polyA-的关键;常规RNA-seq结果;RNA-seq结果：知道链信息的话;dUTP介导的链特异性RNA-seq protocol;DeLi-Seq: Directional Ligation and Sequencing 定向连接介导的RNA测序方法;DeLi-seq能够可靠地检测到裂殖酵母的反义转录体;DeLi-seq能够可靠地检测到裂殖酵母的反义RNA;RNA-seq中基因表达量评估的基本概念;LncRNA的阶段特异性表达;RNA-seq结果;如何测定PolyA+ 和PolyA- lncRNA？;为何区分polyA+和polyA- RNA是重要的;RNA-seq分析流程;实验与生物信息学结合帮助研究生物学问题;举例：环境胁迫对基因表达的影响;RNA-seq的基本分析流程;Nature Protocols, 2012, 7(3):562-578;TopHat网址： /;Tophat的安装;测试TopHat是否安装成功;If TopHat ran successfully, you should see some lines of output, like this: ;Fastq format;测序质量的检查;Genome format (fasta);除Linux外的另一选择：Galaxy;上传原始数据(fastq, or .fq files);View fastq data;上传测试基因组数据(fasta, or .fa);TopHat mapping;Splicing Junction ;RNA analysis menu;课后可对Galaxy功能深入研究;差异表达基因分析;产生bedgraph文件对基因差异表达结果进行可视化验证;Strand-specific RNA-seq track for human T cell;Strand-specific RNA-seq track for SRSF5-overexpression and control in human 293T cell;GO analysis online tools;具体网址;DAVID主页;输入gene symbol;GO分析结果;MSigDB from Broad Institute;输入gene symbol;GO分析结果;Merging sample assemblies with a reference transcriptome annotation;Analyzing groups of transcripts identifies differentially regulated genes;CummeRbund helps users rapidly explore their expression data and create publication-ready plots of differentially expressed and regulated genes;Differential analysis results for regucalcin;Troubleshooting;从头到尾理解转录组;;RT with random primer;Strong location preference;;DeLi-Seq: Directional Ligation and Sequencing 定向连接介导的链特异性RNA-seq方法;3000;Working model;3’-NVTTTdUTTTTTTTTTTTTTTTT-;选择性PolyA加尾对基因表达影响的重要性;PA-seq发现组织特异性选择性polyA加尾;愈加复杂的转录组;Acknowledgements