Pre-mRNA alternative splicing is a prevalent mechanism for diversifying eukaryotic transcriptomes and proteomes. Regulated alternative splicing plays a role in many biological processes, and dysregulated alternative splicing is a feature of many human diseases. Short-read RNA sequencing (RNA-seq) is now the standard approach for transcriptome-wide analysis of alternative splicing. Since 2011, our laboratory has developed and maintained Replicate Multivariate Analysis of Transcript Splicing (rMATS), a computational tool for discovering and quantifying alternative splicing events from RNA-seq data. Here we provide a protocol for the contemporary version of rMATS, rMATS-turbo, a fast and scalable re-implementation that maintains the statistical framework and user interface of the original rMATS software, while incorporating a revamped computational workflow with a substantial improvement in speed and data storage efficiency. The rMATS-turbo software scales up to massive RNA-seq datasets with tens of thousands of samples. To illustrate the utility of rMATS-turbo, we describe two representative application scenarios. First, we describe a broadly applicable two-group comparison to identify differential alternative splicing events between two sample groups, including both annotated and novel alternative splicing events. Second, we describe a quantitative analysis of alternative splicing in a large-scale RNA-seq dataset (~1,000 samples), including the discovery of alternative splicing events associated with distinct cell states. We detail the workflow and features of rMATS-turbo that enable efficient parallel processing and analysis of large-scale RNA-seq datasets on a compute cluster. We anticipate that this protocol will help the broad user base of rMATS-turbo make the best use of this software for studying alternative splicing in diverse biological systems.
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