Computational Comparison of Differential Splicing Tools for Targeted RNA Long-Amplicon Sequencing (rLAS).

RNA sequencing (RNA-Seq) is a powerful technique for the quantification of transcripts and the analysis of alternative splicing. Previously, our laboratory developed the targeted RNA long-amplicon sequencing (rLAS) method, which has the advantage of allowing deep analysis of targeted specific transcripts. The computational tools for analyzing RNA-Seq data have boosted alternative splicing research by detecting and quantifying splicing events. However, the performance of these splicing tools has not yet been investigated for rLAS. Here, we evaluated the performance of four splicing tools (MAJIQ, rMATS, MISO, and SplAdder) using samples with different types of known splicing events (exon-skipping, multiple-exon-skipping, alternative 5' splicing, and alternative 3' splicing). MAJIQ was able to detect all of the types of events, but it was unable to detect one of the exon-skipping events. On the other hand, rMATS was able to detect all of the exon-skipping events. However, rMATS failed to detect other types of events besides exon-skipping events. Both MISO and SplAdder were unable to detect any of the events. These results indicate that MAJIQ presents better performance for the different types of splicing events in rLAS and that rMATS shows better performance for exon-skipping splicing events.