Dividing out quantification uncertainty enables assessment of differential transcript usage with limma and edgeR

Abstract Differential transcript usage (DTU) refers to changes in the relative abundance of transcript isoforms of the same gene between experimental conditions, even when the total expression of the gene doesn’t change. DTU analysis requires the quantification of individual isoforms from RNA-seq data, which has a high level of uncertainty due to transcript overlap and read-to-transcript ambiguity (RTA). Popular DTU analysis methods do not directly account for the RTA overdispersion within their statistical frameworks, leading to reduced statistical power or poor error rate control, particularly in scenarios with small sample sizes. This article presents limma and edgeR analysis pipelines that account for RTA during DTU assessment. Leveraging recent advancements in the limma and edgeR Bioconductor packages, we propose DTU analysis pipelines optimized for small and large datasets with a unified interface via the diffSplice function. The pipelines make use of divided counts to remove RTA-induced dispersion from transcript isoform counts and account for the sparsity in transcript-level counts. Simulations and analysis of real data from mouse mammary epithelial cells demonstrate that the diffSplice pipelines provide greater power, improved efficiency, and improved FDR control compared to existing specialized DTU methods. Competing Interest Statement The authors have declared no competing interest. Footnotes This version includes a more thorough discussion on how to compute TPMs with edgeR and limma fitted values.