Simulation studies to assess the performance of rMATS and the importance of replicates. We simulated 5,000 exons, where 5% of the exons were differentially spliced and the rest were not differentially spliced. We simulated five replicates in each sample group. The exon inclusion levels in individual replicates were simulated from a normal distribution with different SDs in three different studies: (A) SD = 0.01, (B) SD = 0.02, and (C) SD = 0.05. To assess the effect of outliers, we also simulated an additional dataset where 1 of the 10 replicates (of the two sample groups) had a large SD of 0.2. In all scenarios, the analysis by rMATS on replicate data always outperformed the analysis of pooled data (without the information of replicates), as indicated by the receiver operating characteristic (ROC) curves. In addition, the analyses on replicate data were more robust against outliers because rMATS modeled the variation within sample groups, whereas the ROC curves of the pooled data (without the information of replicates) were heavily influenced by outliers.