Relative Abundance of Transcripts (RATs): Identifying differential isoform abundance from RNA-seq

Abstract

The biological importance of changes in RNA expression is reflected by the wide variety of tools available to characterise these changes from RNA-seq data. Several tools exist for detecting differential transcript isoform usage (DTU) from aligned or assembled RNA-seq data, but few exist for DTU detection from alignment-free RNA-seq quantifications. We present the <i>RATs,</i> an R package that identifies DTU transcriptome-wide directly from transcript abundance estimates. <i>RATs</i> is unique in applying bootstrapping to estimate the reliability of detected DTU events and shows good performance at all replication levels (median false positive fraction < 0.05). We compare <i>RATs</i> to two existing DTU tools, <i>DRIM-Seq</i> & <i>SUPPA2,</i> using two publicly available simulated RNA-seq datasets and a published human RNA-seq dataset, in which 248 genes have been previously identified as displaying significant DTU. RATs with default threshold values on the simulated Human data has a sensitivity of 0.55, a Matthews correlation coefficient of 0.71 and a false discovery rate (FDR) of 0.04, outperforming both other tools. Applying the same thresholds for <i>SUPPA2</i> results in a higher sensitivity (0.61) but poorer FDR performance (0.33). RATs and DRIM-seq use different methods for measuring DTU effect-sizes complicating the comparison of results between these tools, however, for a likelihood-ratio threshold of 30, <i>DRIM-Seq</i> has similar FDR performance to <i>RATs</i> (0.06), but worse sensitivity (0.47). These differences persist for the simulated drosophila dataset. On the published human RNA-seq dataset the greatest agreement between the tools tested is 53%, observed between <i>RATs</i> and <i>SUPPA2</i>. The bootstrapping quality filter in <i>RATs</i> is responsible for removing the majority of DTU events called by <i>SUPPA2</i> that are not reported by <i>RATs</i>. All methods, including the previously published qRT-PCR of three of the 248 detected DTU events, were found to be sensitive to annotation differences between Ensembl v60 and v87.