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 transcripts of a gene 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 types of functional annotation, and interoperability with other bioinformatics tools. We showcase how IsoformSwitchAnalyzeR is 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 Alzheimers Disease and single-cell data from Glioblastoma patients. 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.