Development and validation of targeted environmental DNA (eDNA) metabarcoding for early detection of 69 invasive fishes and aquatic invertebrates

Abstract

Invasive species are of concern due to their impacts on ecosystems and economies, but they pose significant control challenges. Environmental DNA (eDNA) is a powerful tool in the detection of aquatic organisms at low densities due to high detection sensitivity and relative ease of sample collection. Aquatic eDNA analyses have increased worldwide and are generally either applied to few target species (quantitative PCR), or for broad taxonomic applications (metabarcoding). Here, we describe the development and testing of a hybrid approach that utilized high‐sensitivity PCR primer sets and high‐throughput sequencing (HTS), referred to as targeted metabarcoding, to detect 69 fishes and invertebrates. We identified target species based on reports of globally important invasive species and developed two independent PCR primers for each species (CO1 and a second mtDNA region). We assessed sensitivity and eDNA interference for all 138 primers (2 per species and 69 species) using standard end‐point PCR and tested them on 10 eDNA samples spiked with various amounts of one or more of the target species DNA. The sensitivity of the 138 primer sets ranged between 1.5 × 10−5 and 2.64 ng template DNA (mean = 0.069 ng). Primers were also tested for interference effects using plankton eDNA to simulate field conditions. The inclusion of interfering plankton DNA reduced the sensitivity for most primer sets by one or more orders of magnitude (range 0–3). Overall, our targeted metabarcoding resulted in the detection of ~98% of species in the DNA spiked samples, and perhaps more importantly, the HTS read count was positively related to the quantity of spiked DNA (p < 0.002). We envision this technique being particularly useful for the early detection of species at low population densities; however, there are diverse applications of targeted metabarcoding for monitoring aquatic community composition and to quantify ecosystem change and health.

Metabarcoding read abundances of orchid mycorrhizal fungi are correlated to copy numbers estimated using ddPCR

“…For example, raw sequencing reads from metabarcoding were transformed into either a presence‐absence metric or normalized metrics of relative abundance data for conducting diversity and community structure analyses (Deagle et al ., 2019). Only a few studies have used other techniques to validate the degree to which read counts accurately reflect real abundances (Wood et al ., 2019; Wu et al ., 2023), leaving the reliability of metabarcoding reads for quantification largely uninvestigated. The present study used ddPCR assays to assess whether metabarcoding reads can be used to quantify diverse mycorrhizal communities that reside in the roots of the terrestrial orchid N. ovata .…”

eDNA Metabarcoding Analysis as Tool to Assess the Presence of Non-Indigenous Species (NIS): A Case Study in the Bilge Water

“…This hybrid methodology is based on the use of metabarcoding and the subsequent design of primer sets to amplify mitochondrial DNA regions of selected species with higher PCR efficiency, so it combines the positive aspects of qPCR and metabarcoding. It was demonstrated to be a potentially valuable tool in invasive species monitoring and detection in aquatic systems [60]. The analyses of eDNA metabarcoding carried out in the bilge waters were able to detect the DNA presence of marine species and specifically of NIS, although they contained concentrated debris that could have complicated laboratory procedures and consequently affected the results.…”