Zebrafish RNA-seq Study Shows Why High Mapping Rates Don't Guarantee Reliable Results

A bioinformatics student built a Nextflow workflow to analyze paired-end RNA sequencing data from zebrafish at the 2-cell and 6-hour developmental stages. The overall read mapping rates were 91.8% and 89.5%, but concordant pair alignment rates differed by 7.4 percentage points, showing that a single mapping percentage does not capture alignment quality. Four gene identifiers passed the Cuffdiff significance threshold, but with only one sample per stage, they are considered exploratory candidates rather than validated findings. The project highlighted that a pipeline can complete successfully without providing enough evidence for strong biological conclusions. The author has shared the workflow, documentation, and limitations on GitHub.