betAS: intuitive analysis and visualisation of differential alternative splicing using beta distributions

Abstract

Next generation RNA sequencing allows alternative splicing (AS) quantification with unprecedented resolution, with the relative inclusion of an alternative sequence in transcripts being commonly quantified by the proportion of reads supporting it as percent spliced-in (PSI). However, PSI values do not incorporate information about precision, proportional to the respective AS events read coverage. Beta distributions are suitable to quantify inclusion levels of alternative sequences, using reads supporting their inclusion and exclusion as surrogates for the two distribution shape parameters. Each such beta distribution has the PSI as its mean value and is narrower when the read coverage is higher, facilitating the interpretability of its precision when plotted. We herein introduce a computational pipeline, based on beta distributions accurately modelling PSI values and their precision, to quantitatively and visually compare AS between groups of samples. Our methodology includes a differential splicing significance metric that compromises the magnitude of inter-group differences, the estimation uncertainty in individual samples, and the intra-group variability, being therefore suitable to multiple-group comparisons. To make our approach accessible and clear to both non-computational and computational biologists, we developed betAS, an interactive web app and user-friendly R package for visual and intuitive differential splicing analysis from read count data.