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Alternative splicing (AS) is an essential co-transcriptional regulatory mechanism in eukaryotes. The accumulation of plant RNA-Seq data provides an unprecedented opportunity to investigate the global landscape of plant AS events. However, most existing AS identification tools were originally designed for animals, and their performance in plants was not rigorously benchmarked. In this work, we developed a simple and easy-to-use bioinformatics tool named ASTool for detecting AS events from plant RNA-Seq data. As an exon-based method, ASTool can detect 4 major AS types, including intron retention (IR), exon skipping (ES), alternative 5′ splice sites (A5SS), and alternative 3′ splice sites (A3SS). Compared with existing tools, ASTool revealed a favorable performance when tested in simulated RNA-Seq data, with both recall and precision values exceeding 95% in most cases. Moreover, ASTool also showed a competitive computational speed and consistent detection results with existing tools when tested in simulated or real plant RNA-Seq data. Considering that IR is the most predominant AS type in plants, ASTool allowed the detection and visualization of novel IR events based on known splice sites. To fully present the functionality of ASTool, we also provided an application example of ASTool in processing real RNA-Seq data of Arabidopsis in response to heat stress.

ASTool: An Easy-to-Use Tool to Accurately Identify Alternative Splicing Events from Plant RNA-Seq Data