From bench to bytes: a practical guide to RNA sequencing data analysis

RNA sequencing (RNA-Seq) is a high-throughput sequencing approach that enables comprehensive quantification of transcriptomes at a genome-wide scale. As a result, RNA-Seq has become a routine component of molecular biology research, and more researchers are now expected to analyze RNA-Seq data as part of their projects. However, unlike the largely experimental nature of benchwork, RNA-Seq analysis demands proficiency with computational and statistical approaches to manage technical issues and large data sizes. Although numerous manuals and reviews on RNA-Seq data analysis are available, many are either highly specialized, fragmented, or overly superficial, leaving beginners to use tools without understanding the underlying principles. To address this gap, we provide a decision-oriented guide tailored for molecular biologists encountering RNA-Seq analysis for the first time. This review is designed for readers to enable to decide which tools and statistical approaches to use based on their data, goals, and constraints. We aim to equip beginners with the knowledge required to perform RNA-Seq analysis rigorously and with confidence.