2.2 Sex determination of harbor seals via scat
To obtain DNA for sex determination, DNA was extracted from the scat matrix-ethanol slurry for all locations, except Cowichan 2014 and Baby Island. For these last two sites, DNA was extracted from the swabs. To extract DNA from swabs, the excess ethanol from the vial was poured off and the swab was dried in a vacuum centrifuge at 39°C until all ethanol had evaporated, approximately one hour. We then used QIAGEN DNeasy Blood and Tissue Kit to extract DNA from the dried swabs. DNA was extracted from slurry matrixes using QIAGEN QIAamp DNA Stool Mini Kit. Extracted DNA, from either the ethanol slurry or swab, was used in Taqman quantitative polymerase chain reactions (qPCR) to determine the presence and absence of X and Y chromosomes. The procedure was modified from Matejusová et al. (2013) and is described in depth in Rothstein (2015). The two probes that we used targeted the paralogous zinc finger X (ZFX) and zinc finger Y (ZFY) genes. Both probes are described in Matejusavá et al. (2013). Two reactions were run for each sample with each probe (four reactions total per sample). Each reaction consisted of: 4.5μl of ABI Taqman gene expression master mix, 0.5μl of either the ZFX or ZFY probe, and 5μl of DNA template. Reactions were run on a quantitative thermocycler with the following protocol: one holding cycle (50°C for 2 min, 95°C for 10 min) followed by 60 cycles of denaturation and annealing/extension (95°C for 15 sec, 60°C for 1 min). Four positive (two reactions for each sex, one ZFX and one ZFY probe each) and four negative controls (two reactions for each ZFX and ZFY probe) were run with each set of reactions. Positive controls came from captive harbor seals of known sex at the Vancouver Aquarium in Vancouver, BC, and Point Defiance Zoo & Aquarium in Tacoma, WA. Negative controls consisted of PCR grade water in place of a DNA template.
If no amplification occurred in either ZFX reactions, the sample was excluded from further analysis. If no amplification occurred in either ZFY reaction, but amplification did occur in either or both reactions with the ZFX probe, the sample was tallied as deposited by a female. If amplification was observed in either or both ZFY reactions, as well as in either or both ZFX reactions, the sample was tallied as deposited by a male. The false negative rate for two failed ZFY reactions (and thereby incorrectly classifying a male as a female) was 1.35%. This value was calculated from the occurrence of only one of the two ZFY reactions having positive amplification within a sample that was classified as male. Although this false negative rate is low, we excluded any samples that amplified in ZFY reactions but failed to amplify in ZFX reactions.