FishMitoCleaner: Automated Stop Codon Processing and Quality Control of Fish Mitochondrial Protein-Coding Sequences

Vertebrate mitochondrial protein-coding genes follow a different genetic code, in which TGA encodes tryptophan and a large number of genes terminate with incomplete stop codons completed post-transcriptionally via polyadenylation. These features are often mishandled when coding sequences (CDS) downloaded from public databases are processed with standard tools, resulting in errors in downstream codon usage and evolution. Here we present FishMitoCleaner, a Python command line tool automating quality control and normalisation of fish mitochondrial CDS sequences in five steps: character validation, internal stop codon scanning, terminal TGA detection, stop codon processing and length normalisation. FishMitoCleaner cleaned 97.6% of input sequences in 22 fish species representing 7 orders and all 13 mitochondrial protein coding genes. Statistical comparison of RSCU values confirmed no bias in codon usage analysis (Welch's t-test, all p > 0.05). FishMitoCleaner is a Python command that allows researchers in aquatic biology and fisheries science to perform rigorous and reproducible CDS quality control in a single command, and clean sequences ready to be analysed in downstream analyses. FishMitoCleaner is also applicable to mitochondrial CDS data from other vertebrate taxa.