GoFish: A Streamlined Environmental DNA Presence/Absence Assay for Marine Vertebrates

Abstract

21Here we describe GoFish, a streamlined environmental DNA (eDNA) presence/absence 22 assay. The assay amplifies a 12S segment with broad-range vertebrate primers, followed by 23 nested PCR with M13-tailed, species-specific primers. Sanger sequencing confirms positives 24 detected by gel electrophoresis. We first obtained 12S sequences from 77 fish specimens 25 representing 36 northwestern Atlantic taxa not well documented in GenBank. Using the newly 26 obtained and published 12S records, we designed GoFish assays for 11 bony fish species 27 common in the lower Hudson River estuary and tested seasonal abundance and habitat 28 preference at two sites. Additional assays detected nine cartilaginous fish species and a marine 29 mammal, bottlenose dolphin, in southern New York Bight. GoFish sensitivity was equivalent to 30 Illumina MiSeq metabarcoding. Unlike quantitative PCR (qPCR), GoFish does not require 31 tissues of target and related species for assay development and a basic thermal cycler is sufficient. 32 Unlike Illumina metabarcoding, indexing and batching samples are unnecessary and advanced 33 bioinformatics expertise is not needed. The assay can be carried out from water collection to 34 result in three days. The main limitations so far are species with shared target sequences and 35 inconsistent amplification of rarer eDNAs. We think this approach will be a useful addition to 36 current eDNA methods when analyzing presence/absence of known species, when turnaround 37 time is important, and in educational settings. 3 38 Introduction 39 DNA profiling of ecological communities was first applied to terrestrial microbes [1]. 40 DNA extracted from soil samples-amplified with ribosomal RNA gene primers, cloned, and 41 analyzed by Sanger sequencing-revealed an enormous diversity of uncultured organisms. 42 Whole genome shotgun sequencing provided an alternative culture-independent approach [2]. 43 Combining targeted amplification with high-throughput sequencing, first 454 then Illumina, 44 eliminated cloning and Sanger sequencing, greatly facilitating microbiome study [3-5]. 45 Around the same time, ancient DNA techniques began to be applied to environmental 46 samples, with recovery of 10,000 years-old to 400,000 years-old plant and animal DNA from 47 fecal samples and sediments [6,7]. The earliest reports examining contemporary materials 48 include differentiating human and domestic sources in sewage-contaminated water [8] and 49 recovery of Arctic fox DNA from snow footprints [9]. Taberlet and colleagues were the first to 50 apply an environmental DNA approach to present-day ecology, demonstrating pond water eDNA 51 accurately surveys an invasive frog species [10]. Subsequent work revealed aquatic eDNA 52 detects diverse vertebrates and invertebrates in multiple habitats [11-15]. Aquatic eDNA assays 53 now routinely monitor rare and invasive freshwater species [16-19]. 54 Beginning in 2003, the DNA barcoding initiative firmly demonstrated that most animal 55 species are distinguished by a short stretch of mito...

In situ Autonomous Acquisition and Preservation of Marine Environmental DNA Using an Autonomous Underwater Vehicle

“…Ship-and shore-based manual sampling methods constrain most field sampling to weekly visitations at best (Bista et al, 2017;Sigsgaard et al, 2017;Stoeckle et al, 2017Stoeckle et al, , 2018. To our knowledge, only a few studies have reported hourly or highfrequency sampling over short durations to investigate the effects tides (Kelly et al, 2018) or spawning events (Tillotson et al, 2018) have on eDNA signals.…”

Species Delimitation of Southeast Pacific Angel Sharks (Squatina spp.) Reveals Hidden Diversity through DNA Barcoding

“…To apply several species delimitation methods to infer MOTUs, our samples were combined with COI sequences from 19 Squatina nominal species (Table 2, Table S1) obtained from the public repository BOLD (www.boldsystems.org, accessed on 5 February 2021), GenBank (www.ncbi.nlm.nih.gov/genbank/, accessed on 5 February 2021), and from the literature [5,35,36,43,[47][48][49][50][51][52][53][54][55][56]. The set of DNA fragments were chosen following two criteria: (1) the location of sampling mentioned in the public database (e.g., country, province, region, sector, exact site, coordinates or FAO fishing zones) and ( 2) length of the DNA fragment (i.e.,~610 bp).…”