Draper Ben J, Dunning Mark J, James David C
Department of Chemical and Biological Engineering, Mappin St., The University of Sheffield, Sheffield, S1 3JD, UK.
Bioinformatics Core Bioinformatics Core, The Faculty of Health,, The University of Sheffield, Sheffield, S10 2HQ, UK.
F1000Res. 2025 May 30;14:47. doi: 10.12688/f1000research.155223.2. eCollection 2025.
Alternative splicing is crucial in gene regulation, with significant implications in clinical settings and biotechnology. This review article compiles bioinformatics short-read RNA-seq tools for investigating differential splicing; offering a detailed examination of their statistical methods, case applications, and benefits. A total of 22 tools are categorised by their statistical family (parametric, non-parametric, and probabilistic) and level of analysis (transcript, exon, and event). The central challenges in quantifying alternative splicing include correct splice site identification and accurate isoform deconvolution of transcripts. Benchmarking studies show no consensus on tool performance, revealing considerable variability across different scenarios. Tools with high citation frequency and continued developer maintenance, such as DEXSeq and rMATS, are recommended for prospective researchers. To aid in tool selection, a guide schematic is proposed based on variations in data input and the required level of analysis. Emerging long-read RNA sequencing technologies are discussed as a complement to short-read methods, promising reduced deconvolution needs and further innovation.
可变剪接在基因调控中至关重要，在临床环境和生物技术领域具有重要意义。这篇综述文章汇编了用于研究差异剪接的生物信息学短读长RNA测序工具；详细介绍了它们的统计方法、案例应用和优势。总共22种工具按其统计类别（参数化、非参数化和概率性）和分析水平（转录本、外显子和事件）进行了分类。量化可变剪接的核心挑战包括正确的剪接位点识别和转录本的准确异构体反卷积。基准研究表明，工具性能尚无共识，不同场景下存在相当大的差异。对于未来的研究人员，推荐使用引用频率高且开发者持续维护的工具，如DEXSeq和rMATS。为了帮助选择工具，基于数据输入的变化和所需的分析水平提出了一个指导示意图。文中还讨论了新兴的长读长RNA测序技术，作为短读长方法的补充，有望减少反卷积需求并推动进一步创新。
