Mitohelper: A mitochondrial reference sequence analysis tool for fish eDNA studies

Abstract

Fish eDNA metabarcoding is a nonintrusive, time- and cost-effective way to detect biodiversity, with potential applications in ecosystem-based management and fisheries assessment. Nevertheless, fish-specific eDNA resources are currently not well-developed, and many fish species are not yet sequenced in reference databases. We developed Mitohelper, a Python-based command line tool to annotate and align fish mitochondrial sequences available in the existing MitoFish database. Mitohelper improves MitoFish annotations by adding gene names and additional taxonomic classifications. Using these improved reference datasets, Mitohelper's getrecord function searches MitoFish for available mitochondrial (cytochrome c oxidase, 12S rRNA, and others) gene sequences against a user-provided list of fish taxonomic names. Mitohelper's getalignment function aligns (often partial) mitochondrial gene sequences to a user-specified full-length reference sequence for the assessment and visualization of overlapping sequencing regions. To facilitate the development of taxonomic classifiers, we combined Mitohelper's 12S rRNA dataset with SILVA’s 16S rRNA and 18S rRNA datasets in a QIIME 2-compatible format. By providing valuable information on taxonomic and gene region coverage of currently available fish mitochondrial data, Mitohelper's functions promote informed experimental design that can guide sequencing and analysis strategies. In summary, Mitohelper improves the breadth and functionality of eDNA data resources, as well as the accuracy of taxonomic classification. Mitohelper and its reference datasets are updated approximately monthly and available at https://github.com/aomlomics/mitohelper.