Fig 1.
Top, schematic of GoFish and MiSeq metabarcoding protocols. Bottom, diagram of 12S and flanking tRNA genes, with locations and sizes of vertebrate metabarcoding targets (MiFish, ECO, Teleo) and the Li segment sequenced from reference specimens as indicated. Typical times for assays are shown; a suitably equipped and staffed laboratory could perform MiSeq metabarcoding in a similar time frame as GoFish.
Fig 2.
Representative GoFish amplifications visualized on 2.5% agarose gel with SYBER Safe.
Lanes bracketed by dates are time series samples from East River site; the last three lanes in each panel are negative controls detailed in Materials and Methods. Marker indicates dye front at approximately 150 bp. Gel positives were sent for Sanger sequencing to confirm target species amplification.
Table 1.
Broad-range vertebrate 12S primers.
PCR parameters and expected amplicon sizes are shown. M13 and Illumina tails in Li primers and ECO V5 primers, respectively, are highlighted in bold.
Table 2.
Species-specific GoFish primers.
Amplicon sizes, PCR parameters, and target specificity as shown. M13 tails are highlighted in bold. All primer sets were newly designed for this study.
Fig 3.
GoFish detections at two lower Hudson estuary locations sampled weekly from March to August 2017.
At top, collection dates are shown; black and white rectangles indicate detection and no detection, respectively, with species arranged by decreasing number of positives; at bottom, number of species detected on each date is shown.
Fig 4.
GoFish detections of cartilaginous fishes and bottlenose dolphin eDNA.
Water samples were collected at one- to two-week intervals from April to December 2017 in southern New York Bight. Black indicates a GoFish detection, white or gray indicates no detection. The gray shading is added to help visualize demarcation of seasons.
Fig 5.
Comparison of GoFish and metabarcoding.
A. Number of detections by method for the 11 species and 34 samples shown in Fig 3. A detection refers to a positive GoFish or metabarcoding result for a single species in a single sample, which corresponds to one cell in Fig 3 grid. Of the 161 GoFish detections shown in Fig 3, 120 were also positive by metabarcoding for the same species in the same sample. In addition, there were 28 metabarcoding detections for one of the 11 target species in samples that were negative by GoFish. B. GoFish detections for the 148 metabarcoding positives, sorted by metabarcoding reads per detection.