Převorovský Martin, Hálová Martina, Abrhámová Kateřina, Libus Jiří, Folk Petr
Department of Cell Biology, Faculty of Science, Charles University, Viničná 7, 128 43 Prague 2, Czech Republic.
Biomed Res Int. 2016;2016:4783841. doi: 10.1155/2016/4783841. Epub 2016 Dec 6.
Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast , about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5' and 3' splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions.
前体mRNA剪接是真核基因表达的一个重要调控层面。在简单的出芽酵母中，所有mRNA分子约有三分之一会经历剪接，并且剪接效率受到严格调控，例如在减数分裂分化过程中。其具有简化的、进化上高度保守的剪接机制，是研究剪接各个方面的理想模型。RNA测序是一种用于转录组分析的强大、通用且经济实惠的技术，也可用于研究剪接效率。然而，缺乏用于分析酵母RNA测序数据中剪接效率的便捷生物信息学工具。我们提出了一个完整的工作流程，用于利用链特异性RNA测序数据计算酵母全基因组的剪接效率。我们的流程以FASTQ格式获取测序读段，并为每个内含子的5'和3'剪接位点提供剪接效率值。该流程基于最新的开源软件工具，对用户输入的要求非常有限。我们以即用型形式提供所有相关脚本。我们使用来自三个剪接体突变体的RNA测序数据集展示了该工作流程的功能。该工作流程对于研究酵母剪接突变体或例如在特定生长条件下的调控剪接应该是有用的。
