Affordable de novo generation of fish mitogenomes using
amplification-free enrichment of mitochondrial DNA and deep sequencing
of long fragments
Abstract
Biomonitoring surveys from environmental DNA 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
long-fragment sequencing approaches using Oxford Nanopore sequencing
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 74.9-805-fold. The targeted
mitosequencing experiment from native genomic DNA yielded 1.83-55 % of
sequences on target and a 38-2123-fold mean coverage. This helped
complete the mitogenome of species with homopolymeric regions, tandem
repeats and gene rearrangements. We demonstrate that deep sequencing of
long fragments of native fish DNA is possible, can be achieved with low
computational resources in a cost-effective manner, exceeding the
widespread genome skimming approach, and allowing the discovery of
mitogenomes of non-model or understudied fish taxa to a broad range of
laboratories worldwide.