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

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 sensitivity and ease of collection. Aquatic eDNA analyses have increased worldwide and are generally either applied to a 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, 69 species) using standard end-point PCR and tested them on 1...