Table 1.
Criteria used for designing primer/probe sets and criteria used to assess primer/probe sets in silico.
Table 2.
Primers and probes designed for target organisms.
Target organism, primer and probe sequences, gene target, fragment size, final concentrations of primers and probe, slope, intercept, limit of quantification, and efficiency of assay.
Fig 1.
Sequence logo plots of in silico sensitivity and specificity of primers and probes for each qPCR assay.
Panel A shows humpback whale, Panel B shows shortbelly rockfish, and Panel C shows common murre. For each panel, the top plots are generated by aligning the regions of the forward primer, reverse primer, and probe, respectively, of target species for each assay and the bottom plots are generated by aligning the same regions for non-target species. The x-axis is the order of base pairs in the primer or probe and the y-axis is the relative frequency of base pair occurrence, with the size of each letter corresponding to its relative frequency. Letters are stacked if multiple base pairs occur at that position in the primer or probe and relative frequencies always sum to 1. The reverse primer for common murre shows as “0” for all base pairs because no contigs were found when non-target species (within the family Alcidae) were aligned with the reverse primer sequence. All sequences are shown from 5’ to 3’.
Table 3.
Sensitivity and specificity of each assay.
Table 4.
Results of in vivo specificity testing.
The three target organisms are shown on the right. Grey shading indicates that the assay was not tested for specificity using the organism on in the first column. “NA” indicates no amplification (i.e., assay is specific). For the non-target species that amplified, the Cq values are shown for each individual. 1–2 ng of gDNA was included in each reaction; see Fig 2 for Cq values for target individuals.
Fig 2.
Cycle quantification threshold (Cq) is shown on the y-axis and the x-axis is the concentration of DNA (pg gDNA/μL) in the reaction; 2 μL of template was added to each reaction for the dilution series. Panel A shows humpback whale (Megaptera novaeangliae), panel B shows common murre (Uria aalge), and panel C shows shortbelly rockfish (Sebastes jordani). For each plot, black crosses are the results of the individuals used for specificity testing (C551 for humpback whale, 06–0175 for common murre, 4450 for shortbelly rockfish). Blue x’s are the results of the individuals used for environmental sample testing (C500 for humpback whale, 06–0172 for common murre, RCSB3 for shortbelly rockfish). In the case of shortbelly rockfish (Panel C), some of the specificity testing was also performed using the standard curve used for environmental sample testing. Each concentration was run in triplicate reactions and in some cases, symbols are overlapping.
Table 5.
Results of environmental samples tested for each assay.
± is 95% confidence interval. “BLOQ” represents below the limit of quantification. “ND” represents non-detect, meaning sample was not assigned a Cq value.