Abstract
Alternative pre-mRNA splicing (AS) is a fundamental regulatory process that generates transcript diversity and cell type variation. We developed Shiba, a comprehensive method that integrates transcript assembly, splicing event identification, read counting, and differential splicing analysis across RNA-seq platforms. Shiba excels in capturing annotated and unannotated AS events with superior accuracy, sensitivity, and reproducibility. It addresses the often-overlooked issue of junction read imbalance, significantly reducing false positives to aid target prioritization and downstream analyses. Unlike other tools that require large numbers of biological replicates or resulting in low sensitivity and high false positives, Shiba's statistics framework is agnostic to sample size, as demonstrated by simulated data and its effective application to real n=1 RNA-seq datasets. To extend its utility to single-cell RNA-seq, we developed scShiba, which applies Shiba's pseudobulk approach to analyze splicing at the cluster level. scShiba successfully revealed AS regulation in developmental dopaminergic neurons and differences between excitatory and inhibitory neurons. Both Shiba and scShiba are available in Docker/Singularity containers and Snakemake pipelines, ensuring reproducibility. With their comprehensive capabilities, Shiba and scShiba enable systematic quantification of alternative splicing events across various platforms, laying a solid foundation for mechanistic exploration of the functional complexity in RNA splicing.