A re-analysis of an existing Drosophila melanogaster dataset reveals a new set of genes involved in post-mating response

The figures and tables presented here are part of a manuscript submitted for publication by Chloe J. Bennett and Rodolfo Aramayo entitled: "A re-Analysis of an existing Drosophila melanogaster dataset 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.本文呈现的图表与表格，均属于Chloe J. Bennett与Rodolfo Aramayo提交待发表的手稿，题为"对现有黑腹果蝇（Drosophila melanogaster）数据集的再分析揭示了一组参与交配后应答的新基因"。 摘要 RNA测序（RNA-seq）是识别两种条件下基因表达差异的常用手段。但RNA-seq产出数据的分析流程复杂，相同原始数据可能得到不同分析结果。为此，我们开发并部署了四套并行分析流程，对交配前后的两种雌性黑腹果蝇组织的现有数据集进行再分析。黑腹果蝇的交配后应答（post-mating response, PMR）是一套已被充分表征的交配后生理变化过程，伴随基因表达的动态波动。 将本研究与该数据集的既往分析结果对比后发现，本研究的分析结果更为严谨，同时还鉴定出了一批既往研究未发现的显著差异表达基因。此外，我们还观察到自身独立实验间存在差异，基因-转录本异构体数量及索引构建流程对分析结果具有重要影响。最终，我们通过自研流程鉴定出了一批未被既往研究发现的基因，并提出了这些基因在交配后生物学过程中的潜在功能。综上，本研究对当前RNA-seq分析技术进行了批判性评估，并提出了多项流程优化方案，可提升差异基因表达研究的灵敏度、特异性与严谨性。