A Re-Analysis of an ExistingDrosophila melanogasterDataset Reveals a New Set of Genes Involved in Post-Mating Response

ABSTRACT RNA sequencing (RNA-seq) is a commonly used method to identify changes in gene expression between two conditions. The analysis of RNA-seq output is complicated, with the possibility of getting different results from the same raw data. We developed and deployed four parallel pipelines to reanalyze an existing dataset of two female Drosophila melanogaster tissue types before and after mating. The Drosophila post-mating response (PMR) is a well-characterized suite of changes that occur after mating, accompanied by a flux in gene expression. In comparing our study with the previous analysis of this dataset, we find our results to be more stringent, though we do identify a number of significant genes not found before. We also found variation among our own separate experiments, with gene-to-transcript isoform number and index building playing important roles in outcome. Finally, we identified a set of genes found by our pipeline that were not identified by the previous study and proposed potential roles for these genes in post-mating biology. Together, this work presents a critique of current RNA-seq analysis techniques and proposes multiple workflow adjustments that can increase the sensitivity, specificity, and stringency of differential gene expression studies.