A Dual Approach to Evaluate the Performance of RNA-Seq Data Analysis Pipelines with Weak Signals

RNA-Seq data analysis involves several critical steps, from quality control to differential expression analysis, each offering multiple algorithmic options. Selecting the optimal pipeline is particularly challenging in complex scenarios with weak signals.This study highlights the significant impact of pipeline selection on RNA-Seq data analysis, particularly in contexts characterized by weak transcriptional signals, as in this study for low-dose metallic particle exposure. By evaluating 90 bioinformatics pipelines using two complementary approaches — correlation with qRT-PCR data and classification performance — we were able to identify optimal configurations that ensure both analysis robustness for biological relevant observation.