The utmost goal of selecting an RNA-Seq alignment software is to perform accurate alignments with a robust algorithm, which is capable of detecting the various intricacies underlying read-mapping procedures and beyond. Most alignment software tools are typically pre-tuned with human or prokaryotic data, and therefore may not be suitable for applications to other organisms, such as plants. The rapidly growing plant RNA-Seq databases call for the assessment of the alignment tools on curated plant data, which will aid the calibration of these tools for applications to plant transcriptomic data. We therefore focused here on benchmarking RNA-Seq read alignment tools, using simulated data derived from the model organism Arabidopsis thaliana. We assessed the performance of five popular RNA-Seq alignment tools that are currently available, based on their usage (citation count). By introducing annotated single nucleotide polymorphisms (SNPs) from The Arabidopsis Information Resource (TAIR), we recorded alignment accuracy at both base-level and junction base-level resolutions for each alignment tool. In addition to assessing the performance of the alignment tools at their default settings, accuracies were also recorded by varying the values of numerous parameters, including the confidence threshold and the level of SNP introduction. The performances of the aligners were found consistent under various testing conditions at the base-level accuracy; however, the junction base-level assessment produced varying results depending upon the applied algorithm. At the read base-level assessment, the overall performance of the aligner STAR was superior to other aligners, with the overall accuracy reaching over 90% under different test conditions. On the other hand, at the junction base-level assessment, SubRead emerged as the most promising aligner, with an overall accuracy over 80% under most test conditions.
Keywords: Arabidopsis thaliana; RNA-Seq; SNPs; alignment tools; benchmarking; read alignment; transcriptomics.