Incorporating Exon–exon Junction Reads Enhances Differential Splicing Detection

RNA sequencing (RNA-seq) is a gold standard technology for studying gene and transcript expression. Different transcripts from the same gene are usually determined by varying combinations of exons within the gene, formed by splicing events. One method of studying differential alternative splicing between groups in short-read RNA-seq experiments is through differential exon usage (DEU) analysis, which uses exon-level read counts along with downstream statistical testing strategies. However, the standard exon counting method does not consider exon-junction information, which may reduce the statistical power in detecting splicing alterations. We present a new workflow for differential splicing analysis, called differential exon-junction usage (DEJU). This DEJU analysis workflow adopts a new feature quantification approach that jointly summarises exon and exon–exon junction reads, which are then integrated into the established Rsubread-edgeR/limma frameworks. We performed comprehensive simulation studies to benchmark the performance of DEJU against existing methods. We also applied DEJU to a mouse mammary gland RNA-seq dataset, revealing biologically meaningful splicing events that could not be detected previously. We demonstrate that incorporating exon–exon junction reads significantly improves the detection of differential splicing events. The proposed DEJU workflow offers increased statistical power and computational efficiency compared to widely used existing approaches, while effectively controlling the false discovery rate.