Affordable de novo generation of fish mitogenomes using amplification-free enrichment of mitochondrial DNA and deep sequencing of long fragments.

Biomonitoring surveys make use of metabarcoding tools to describe the community composition. These studies match their sequencing results against public genomic databases to identify the species. However, mitochondrial genomic reference data are yet incomplete, only a few genes may be available, or the suitability of existing sequence data is suboptimal for species level resolution. Here, we present a dedicated and cost-effective workflow with no DNA amplification for generating complete fish mitogenomes for the purpose of strengthening fish mitochondrial databases. Two different strategies using long fragment sequencing with Oxford Nanopore technology coupled with mitochondrial DNA enrichment were used. One where the enrichment is achieved by preferential isolation of mitochondria followed by DNA extraction and nuclear DNA depletion ("mitoenrichment"). A second enrichment approach takes advantage of the CRISPR Cas9 targeted scission on previously dephosphorylated DNA ("targeted mitosequencing"). The sequencing results varied between tissue, species, and integrity of the DNA. The mitoenrichment method yielded 0.17%-12.33% of sequences on target and a mean coverage ranging from 74.9 to 805-fold. The targeted mitosequencing experiment from native genomic DNA yielded 1.83%-55% of sequences on target and a 38 to 2123-fold mean coverage. These produced complete mitogenomes of species with homopolymeric regions, tandem repeats, and gene rearrangements. We demonstrate that deep sequencing of long fragments of native fish DNA can be achieved with low computational resources in a cost-effective manner, opening the discovery of mitogenomes of nonmodel or understudied fish taxa to a broad range of laboratories worldwide.