GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates

Abstract

Here we describe GoFish, a strategy for single-species environmental DNA (eDNA) presence/absence assays using nested PCR. The assays amplify a mitochondrial 12S rDNA segment with vertebrate metabarcoding primers, followed by nested PCR with M13-tailed, species-specific primers. Sanger sequencing confirms positives detected by gel electrophoresis. We first obtained 12S sequences from 77 fish specimens for 36 northwestern Atlantic taxa not well documented in GenBank. Using these and existing 12S records, we designed GoFish assays for 11 bony fish species common in the lower Hudson River estuary and tested seasonal abundance and habitat preference at two sites. Additional assays detected nine cartilaginous fish species and a marine mammal, bottlenose dolphin, in southern New York Bight. GoFish sensitivity was equivalent to Illumina MiSeq metabarcoding. Unlike quantitative PCR (qPCR), GoFish does not require tissues of target and related species for assay development and a basic thermal cycler is sufficient. Unlike Illumina metabarcoding, indexing and batching samples are unnecessary and advanced bioinformatics expertise is not needed. From water collection to Sanger sequencing results, the assay can be carried out in three days. The main limitations to this approach, which employs metabarcoding primers, are the same as for metabarcoding, namely, inability to distinguish species with shared target sequences and inconsistent amplification of rarer eDNA. In addition, the performance of the 20 assays reported here as compared to other single-species eDNA assays is not known. This approach will be a useful addition to current eDNA methods when analyzing presence/absence of known species, when turnaround time is important, and in educational settings.

Novel universal primers for metabarcoding environmental DNA surveys of marine mammals and other marine vertebrates

“…Finally, the high levels of diagnostic variation seen within MarVer loci amplicons offer potential for designing additional species-specific nested internal primers (Stoeckle, Das, & Charlop-Powers, 2018).…”

Application of Environmental DNA Metabarcoding to Spatiotemporal Finfish Community Assessment in a Temperate Embayment

“…Seventeen major and 25 rare finfish ASVs were identified during summer 2017. While this study benefited greatly from recent contribution of local reference sequences into GenBank (Stoeckle et al, 2018), and by validation of eDNA identification using historical DEEP trawl survey data, we suggest that the incomplete status of reference sequence databases for finfish species remains an important research gap. To improve reference databases, we recommend: (1) contributing known species sequences currently absent, especially those that are locally relevant; (2) updating low-quality sequences; and (3) incorporating multi-metabarcoding when single metabarcode does not provide definitive taxonomic information.…”

“…For instance, three rare ASVs were identified as goldtail angelfish (Pomacanthus chrysuru), unicorn leatherjacket (Aluterus monoceros),and ocellated flounder (Ancylopsetta quadrocellata) at 96, 91, and 97%, when classification was first done in 2018, prompting the conclusion that these rare ASVs represented finfish taxa that did not yet have reference sequences available. As reference sequences of under-documented northwestern Atlantic finfish became available in GenBank (Stoeckle et al, 2018), the 3 rare ASVs were re-classified as striped cusk-eel (Ophidion marginatum), northern stargazer (Astroscopus guttatus), and fourspot flounder (Paralichthys oblonga) with 99, 100, and 100% match. These examples demonstrate the value of acquiring reference sequences from species with currently low GenBank representation.…”

MiFish metabarcoding: a high-throughput approach for simultaneous detection of multiple fish species from environmental DNA and other samples

“…The versatility of MiFish primers for gene amplification across diverse taxa has led to the development of two new techniques for single-species detections (Stoeckle et al 2018;Truelove et al 2019). In addition, two new software tools have been developed for bioinformatics pipeline (Curd et al 2019) and ecological analyses based on an output taxonomic table (Kandlikar et al 2018), with their own analyzed data from MiFish eDNA metabarcoding as examples to demonstrate software performance.…”