Abstract Background Current RNA-seq analysis software for RNA-seq data tends to use similar parameters across different species without considering species-specific differences. However, the suitability and accuracy of these tools may vary when analyzing data from different species, such as humans, animals, plants, fungi, and bacteria. For most laboratory researchers lacking a background in information science, determining how to construct an analysis workflow that meets their specific needs from the array of complex analytical tools available poses a significant challenge. Results By utilizing RNA-seq data from plants, animals, and fungi, it was observed that different analytical tools demonstrate some variations in performance when applied to different species. A comprehensive experiment was conducted specifically for analyzing plant pathogenic fungal data, focusing on differential gene analysis as the ultimate goal. In this study, 288 pipelines using different tools were applied to analyze five fungal RNA-seq datasets, and the performance of their results was evaluated based on simulation. This led to the establishment of a relatively universal and superior fungal RNA-seq analysis pipeline that can serve as a reference, and certain standards for selecting analysis tools were derived for reference. Additionally, we compared various tools for alternative splicing analysis. The results based on simulated data indicated that rMATS remained the optimal choice, although consideration could be given to supplementing with tools such as SpliceWiz. Conclusion The experimental results demonstrate that, in comparison to the default software parameter configurations, the analysis combination results after tuning can provide more accurate biological insights. It is beneficial to carefully select suitable analysis software based on the data, rather than indiscriminately choosing tools, in order to achieve high-quality analysis results more efficiently.

A comprehensive workflow for optimizing RNA-seq data analysis