In silico assessment of primers for eDNA studies using PrimerTree and application to characterize the biodiversity surrounding the Cuyahoga River

Abstract

Analysis of environmental DNA (eDNA) enables the detection of species of interest from water and soil samples, typically using species-specific PCR. Here, we describe a method to characterize the biodiversity of a given environment by amplifying eDNA using primer pairs targeting a wide range of taxa and high-throughput sequencing for species identification. We tested this approach on 91 water samples of 40 mL collected along the Cuyahoga River (Ohio, USA). We amplified eDNA using 12 primer pairs targeting mammals, fish, amphibians, birds, bryophytes, arthropods, copepods, plants and several microorganism taxa and sequenced all PCR products simultaneously by high-throughput sequencing. Overall, we identified DNA sequences from 15 species of fish, 17 species of mammals, 8 species of birds, 15 species of arthropods, one turtle and one salamander. Interestingly, in addition to aquatic and semi-aquatic animals, we identified DNA from terrestrial species that live near the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that had not been previously reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from small water samples using wide-range PCR amplification combined with high-throughput sequencing can provide a broad perspective on biological diversity.

Demonstration of the potential of environmental DNA as a tool for the detection of avian species

“…In addition, inter-specific differences in the edit distance were calculated and 82,177 out of 82,621 combinations (99.5%) showed edit distance larger than 5 (Table 4). To examine the range of species that can be amplified using MiBird-U primers, we performed an analysis with the primerTree package [25]. The results confirmed that the primers can amplify avian species (Fig.1b).…”

“…Thus, by using primerTree package, species whose sequences can be amplified, phylogenetic relationships among these amplified species, and interspecific variations in the amplified sequences are rapidly visualized. Further information and instructions for the primerTree package can be found in Cannon et al [25] .…”

“…In addition, the binding capacity and the levels of interspecific variations of the target region were further evaluated using 'primerTree' package [25] of R version 3.3.1 [26]. Briefly, primerTree performs the following analysis: (1) In silico PCR against sequences in the NCBI database; (2) retrieval of DNA sequences predicted to be amplified; (3) taxonomic identification of these sequences; (4) multiple DNA sequence alignment; (5) reconstruction of a phylogenetic tree and (6) visualization of the tree with taxonomic annotation.…”

Toward an ecoregion scale evaluation of eDNA metabarcoding primers: A case study for the freshwater fish biodiversity of the Murray–Darling Basin (Australia)

“…In addition to these trade‐offs, species biodiversity varies extensively between geographic regions (Abell et al., ; Olson et al., ; Spalding et al., ) which further complicates the selection process as the performance of metabarcoding primers will vary depending on the species composition at the sampling location. In recent years, a number of software programs have been developed for the in silico evaluation of metabarcoding primers (Boyer et al., ; Cannon et al., ; Elbrecht & Leese, ; Ficetola et al., ; Foster, Sharpton, & Grünwald, ; Riaz et al., ). However, most studies to date have only evaluated primers using a single program, and the performance of primer performance has not been evaluated at a regional scale.…”

Metabarcoding of Environmental DNA Samples to Explore the Use of Uranium Mine Containment Ponds as a Water Source for Wildlife

“…No other DNA from tissue extractions used in primer development were identified in our runs. We cannot account for the chicken, turkey or pig sequences contained in our runs; however, other studies have reported reads from these species as well as from cattle and suggest that these reads may have come from animal feed found in aquaria from which samples were taken [25], fecal matter from livestock swept down river [51] or contaminated PCR reagents, particularly dNTPs [35]. We believe that most of the chicken, turkey and pig sequence could have come from our PCR reagents, but further investigation is required.…”