Environmental DNA Metabarcoding Assay Selection, Sequencing Depth, and PCR Stochasticity Affects Detection of Amphibian Communities
Amphibian populations are declining on a global scale, necessitating methods to facilitate robust long-term monitoring to support conservation efforts. Environmental DNA (eDNA) metabarcoding can detect the amphibian species present within a waterbody if the assay is well designed, validated, and applied alongside an appropriate sampling design. However, a standardized workflow to select and optimize amphibian metabarcoding assays is lacking. The goal of this study was to explore the importance of an explicit amphibian eDNA metabarcoding assay selection and optimization workflow to better inform practices using the amphibian community of the Grand River watershed in Ontario, Canada, as a case study. After in silico evaluation of 11 published eDNA metabarcoding assays targeting mitochondrial ribosomal RNA genes, most published assays did not meet defined in silico criteria to characterize the target amphibian community. After in vitro optimization of the selected mt16S and de novo mt12S metabarcoding assays, eDNA extracted from water samples gathered from multiple vernal pools was used for in situ evaluation, enabling iterative improvement of the mt12S assay. For each assay, the effect of technical replicate number and read depth on amphibian species detections were evaluated. While each assay detected amphibian eDNA, the mt12S assay detected the most amphibian species with fewer non-target detections. Detection of amphibian species improved with increased technical replicates and read depths. These results emphasize the importance of careful evaluation and optimization of assay conditions for local target and non-target taxa when implementing amphibian eDNA monitoring programs.