Novel Universal Primers for Metabarcoding eDNA Surveys of Marine Mammals and Other Marine Vertebrates

Abstract

Metabarcoding studies using environmental DNA (eDNA) and high throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals have received less attention. Similarly, there have been few attempts to identify potentially ‘universal’ barcode loci which may be informative across multiple marine vertebrate Orders. Here we describe the design and validation of four new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. ‘In silico’ validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators.

Improving metabarcoding taxonomic assignment: A case study of fishes in a large marine ecosystem

“…Because of the high degree of similarity between the MiFish Universal and Elasmobranch loci and the flexibility built into CRUX, a single CRUX generated 12S reference database performs well for both markers (Curd et al, 2019), so we did not create separate teleost and elasmobranch databases. Additionally, because the MiFish primer set amplifies nearly all vertebrate taxa (Miya et al, 2015;Valsecchi et al, 2019), the global database include teleosts, elasmobranchs, mammals, reptiles, amphibians, birds, etc. All databases are available at https://doi.org/10.5068/D1H963.…”

“…This result stems from the regional database being specific to California Current Large Marine Ecosystem fishes, and could thus not identify a marine mammal. However, this shortcoming could easily be overcome by appending the regional database with barcodes for other marine-associated vertebrate taxa of regional management interests (Valsecchi et al, 2019). An alternative and taxon agnostic approach currently employed by the coauthors is to F I G U R E 3 Effect of bootstrap confidence cutoff scores on taxonomic assignment metrics.…”

“…In the design of the MiFish Teleost 12S primers, uncultured marine microbe 16S sequences were not considered as potential alternative targets for the primer set, resulting in the selection of a metabarcoding locus with a high degree of nontarget amplification (Miya et al, 2015). This finding is important for the marine vertebrate eDNA community, which has recently converged on the MiFish 12S primers as the vertebrate barcode of choice (Closek et al, 2019;Miya et al, 2020;O'Donnell et al, 2017;Valsecchi et al, 2019;Yamahara et al, 2019). At best, this nontarget amplification of microbial DNA will lead to wasted sequencing effort, as every microbial sequence generated reduces the number of vertebrate sequences captured.…”

“…The MiFish Universal Teleost and MiFish Elasmobranch primer sets (Miya et al, 2015) target the same portions of the mitochondrial 12S RNA gene, but differ by a few critical base pairs on the forward primer. These metabarcodes are vertebrate specific, provide species‐level resolution for many fishes, and are well suited to short read‐length next‐generation DNA sequencing, such as Illumina platforms (Collins et al, 2019; Jo et al, 2017; Miya et al, 2015; Valsecchi et al, 2019). As such, they are becoming the standard barcode locus for marine vertebrate metabarcoding studies (Bista et al, 2017; Closek et al, 2019; Miya et al, 2015; Thomsen et al, 2016; Valsecchi et al, 2019; Yamamoto et al, 2017).…”

“…These metabarcodes are vertebrate specific, provide species‐level resolution for many fishes, and are well suited to short read‐length next‐generation DNA sequencing, such as Illumina platforms (Collins et al, 2019; Jo et al, 2017; Miya et al, 2015; Valsecchi et al, 2019). As such, they are becoming the standard barcode locus for marine vertebrate metabarcoding studies (Bista et al, 2017; Closek et al, 2019; Miya et al, 2015; Thomsen et al, 2016; Valsecchi et al, 2019; Yamamoto et al, 2017). However, 12S fish reference databases are relatively incomplete compared to traditional barcoding loci, such as the 655 bp region of the mitochondrial cytochrome oxidase I ( COI ) gene (Ardura et al, 2013; Duke & Burton, 2020; Hastings & Burton, 2008; Ward et al, 2009).…”