A practical and reproducible workflow for validation and characterization of animal mitochondrial genomes: from raw sequencing reads and GenBank records to publication-ready mitogenomic analyses

Mitochondrial genomes are widely used in zoology for taxonomy, systematics, phylogeography, biodiversity assessment and comparative genomics, yet the steps required to validate and characterize them are scattered across programs and methodological traditions. This is a particular problem for researchers beginning mitogenomic studies, because automated assemblies and annotations may contain incomplete genes, incorrect gene boundaries, undetected tRNAs, unresolved repetitive regions or unsupported claims of circularization. Here, a practical, step-by-step workflow is presented for animal mitochondrial genomes. It can be applied either to raw paired-end sequencing reads together with an assembled mitogenome (Workflow A) or to previously assembled mitogenomes obtained from public databases such as GenBank (Workflow B). The workflow integrates read quality control, read mapping, genome-wide and gene-level coverage assessment, annotation auditing, validation of open reading frames, tRNA reassessment, direct testing of circularization, nucleotide composition and compositional skews, protein-coding gene characterization, codon usage, relative synonymous codon usage, amino-acid composition, rRNA characterization, gene order, intergenic spacers, overlaps and non-coding regions. It explicitly separates analyses that require original sequencing reads from those that can be performed with annotated sequences alone, and it provides conservative interpretation rules for common pitfalls, such as treating a gene that was not detected as a gene that was lost, or coding completeness as proof of circularization. Every step is accompanied by generalized, commented command-line and Python examples in the Supplementary Material. The framework emphasizes traceability, preservation of intermediate outputs and a clear distinction between biological variation and annotation or assembly artifacts. It is intended as an accessible entry point for researchers beginning mitogenomic studies and as a reproducible framework for producing publication-ready descriptions of animal mitochondrial genomes. “Supplementary Material and the script 06b_junction_spanning_reads.py are included in this record.”