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.

A CRISPR-based strategy for targeted sequencing in biodiversity science

“…Although CRISPR is generally underutilized in environmental science (Phelps et al, 2020), CRISPR-based enrichment strategies have shown promise and versatility (Baerwald et al, 2023;López-Girona et al, 2020;Ramon-Laca et al, 2022;Sánchez et al, 2022;Sandoval-Quintana et al, 2023;Williams et al, 2023). We evaluated strategies to harness this power for important applications such as overcoming the plant DNA barcode resolution problem (CBOL Plant Working Group, 2009;Kress, 2017) and issues with PCR-based DNA relative read abundance calculations (Deagle et al, 2019;Littleford-Colquhoun, Freeman, et al, 2022).…”

“…For instance, while many previous studies have shown some degree of off-target enrichment when using single-species samples (López-Girona et al, 2020;Ramon-Laca et al, 2022), Sandoval-Quintana et al (2023) found that only 0.03% of good quality reads covered the region they wished to study when enriching a bacterial gene from a complex microbial sample. To address this challenge, it may be useful to experiment with mismatch tolerance values.…”

“…Our in-silico analysis of plant gRNAs coupled with our six validation experiments provide a proof of concept involving the use of CRISPR-based enrichment sequencing for use in environmental biology. This technology can be used to build accurate plant DNA barcode libraries with sequences that are long enough to span multiple barcode regions and thus overcome long-standing limitations to taxonomic resolution in PCR-based barcoding studies (CBOL Plant Working Group, 2009;Kress, 2017)-potentially providing sequences for entire chloroplast genomes-though overcoming the challenge of translating this potential into versatile and cost-effective methods for analysis of environmental DNA represents an exciting area for development (Ramon-Laca et al, 2022;Schultzhaus et al, 2021). Moving forward, these approaches can be extended to incorporate other facets of research that are integral for biodiversity discovery, such as determining structural rearrangements (Sun et al, 2022), phylogenetic patterns (Yang et al, 2014), targeted genome sequencing (López-Girona et al, 2020), multiplexing samples and loci within a single reaction, and building metagenome-assembled genomes (Liu et al, 2022;Sandoval-Quintana et al, 2023).…”

“…When similar approaches are applied to samples containing mixtures of DNA from multiple species using DNA metabarcoding, reliance on PCR involves further challenges associated with detecting and estimating the relative abundance of phylogenetically disparate taxa (Clarke et al, 2014;Deagle et al, 2019;Kelly et al, 2019;O'Donnell et al, 2016;Stapleton et al, 2022). By contrast, CRISPR-Cas technology may enable researchers to circumvent several of these challenges: it has recently enabled the targeted enrichment of entire mitogenomes from diverse fishes (Ramon-Laca et al, 2022); it has enabled the detection of specific DNA strands in environmental DNA (Baerwald et al, 2023;Karlikow et al, 2023;Sánchez et al, 2022;Williams et al, 2023;Williams et al, 2021;Williams et al, 2019); and it has revealed both single-nucleotide and structural variants in the locus controlling for fruit color in apples (López-Girona et al, 2020). Yet despite the potential to use CRISPR-Cas to overcome drawbacks to PCR by providing longer, and hence more diagnostic markers, this strategy has not yet been tested in comparative analyses involving multiple target sequences.…”

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

“…Raw sequencing data have been made available on Dryad, data set (Ramón-Laca et al, 2022), https://doi.org/10.5061/dryad.jm63x sjdj.…”