IsoformSwitchAnalyzeR v2: analysis of functional isoform changes in long-read and single-cell sequencing data.

Alternative splicing enables a single gene to produce a variety of mRNA transcripts, significantly enhancing protein diversity in higher eukaryotes. Isoform switching refers to the differential usage of a gene's transcripts and occurs pervasively across physiological and pathological conditions. IsoformSwitchAnalyzeR was developed to identify these isoform switches and analyze their functional consequences. Advances in RNA-seq technology, including long-read and single-cell sequencing, along with state-of-the-art computational tools, enable unprecedented accuracy in isoform switch identification and its functional consequences, necessitating an update to IsoformSwitchAnalyzeR. Here, we present IsoformSwitchAnalyzeR 2.0, with substantial improvements in the robustness of isoform switch detection, the incorporation of new functional annotation types, and interoperability with other bioinformatics tools. We showcase how IsoformSwitchAnalyzeR's standard workflow is now well-suited for analysis of both long-read RNA-seq and single-cell data through two case studies. Specifically, we analyze long-read data from patients with Alzheimer's disease and single-cell data from patients with glioblastoma. In both case studies, we find important isoform switches with disease-relevant functional consequences, showcasing the power of IsoformSwitchAnalyzeR v2. Taken together, these findings highlight the versatility and robustness of IsoformSwitchAnalyzeR in handling advanced sequencing technologies, thereby broadening its applicability across diverse research contexts.