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

Abstract

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.

A CRISPR‐based strategy for targeted sequencing in biodiversity science

“…In Experiment 2, however, we had mixed success with a tiling approach because only one of the tiled gRNAs was effec- for analysis of environmental DNA represents an exciting area for development (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 (Li et al, 2016;Ramón-Laca et al, 2023;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 (Stangl et al, 2020;Welch et al, 2022), and building metagenome-assembled genomes (Liu et al, 2022;Sandoval-Quintana et al, 2023).…”

“…Although CRISPR is generally underutilized in the environmental sciences (Phelps et al., 2020), CRISPR‐based enrichment strategies have shown promise and versatility (Baerwald et al., 2023; López‐Girona et al., 2020; Ramón‐Laca et al., 2023; Sánchez et al., 2022; Sandoval‐Quintana et al., 2023; Stangl et al., 2020; Williams et al., 2023). We evaluated strategies to harness this power for important applications in environmental biology 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).…”

“…By contrast, CRISPR-Cas systems may enable researchers to circumvent several of these challenges and overcome drawbacks to PCR by providing longer and hence more diagnostic markers. Recent CRISPR applications in environmental biology have already exemplified its versatility by detecting specific DNA strands in environmental DNA (Baerwald et al, 2023;Karlikow et al, 2023;Sánchez et al, 2022;Shashank et al, 2023;Williams et al, 2019Williams et al, , 2021Williams et al, , 2023, enabling the targeted enrichment of fish mitogenomes (Ramón-Laca et al, 2023), and identifying structural variants in loci controlling for colour in apples (López-Girona et al, 2020). However, it has not yet been used to study the chloroplast genomes of plants or used in comparative studies involving multiple target sequences within a single sample.…”

“…All target taxa were detected with CRISPR-Cas enrichment which produced contig lengths per taxon that were at least 48-fold longer than amplicon sequencing.may have contributed to non-target enrichment in Experiment 1, where we found a de novo contig that mapped upstream of the target region and in Experiments 5 and 6 where non-chloroplast contigs were generated. While many previous studies have shown some degree of off-target enrichment when using single-species samples(López-Girona et al, 2020;Ramón-Laca et al, 2023), 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, indicat-…”

rCRUX: A rapid and versatile tool for generating metabarcoding reference libraries in R

“…However, the utility of rCRUX allows for reference databases to be generated on any blast-formatted database, directly supporting improved taxonomic assignment of a broad range of DNA sequence applications. For example, this allows for the curation of reference barcodes from full-or partiallength mitogenomes (see Data S1), supporting long-read sequencing taxonomic assignment applications (Johri et al, 2019;Ramon-Laca et al 2022). In addition, rCRUX can be used for building nuclear DNA-based reference databases from whole-genome and transcriptome sequences.…”