Genotyping Success
After removing individuals with >50% missing data, a total of 365 samples, including 3 duplicate samples, remained in our GT-seq dataset. Average missing data dropped to 3.2% from 21.3% when these individuals were removed. Percent individuals successfully genotyped ranged from 30.6% to 97.4% depending on sample type (Table 1), recalling that most of the FF samples were not screened before sequencing to allow for testing of the qPCR screening tool (Hayward et al., 2020). When considering only the FF samples that would have passed screening (>0 ng/µL polar bear DNA), percent individuals successfully genotyped increases to 85.7%. See Supplemental Information: qPCR Experiment for detailed results.
For retained individuals with >50% data, average missing data per locus was similar across sample types, with FF samples missing the most at 14.9% of loci (Table 1). Both BP replicates were successfully genotyped at >50% loci and gave a mean genotyping error rate of 0.17%. Of the 21 FF samples for which duplicate subsamples were included in the GT-seq runs without qPCR pre-screening, one or both subsamples failed to amplify in 20 of 21 cases. In most cases (15/21), both subsamples failed to amplify, although there were a few cases where we witnessed large differences in genotyping success between FF duplicates. For example, one FF subsample was genotyped at only 8 loci, whereas its corresponding subsample was genotyped at all 322 loci. For the one FF sample for which both subsamples successfully genotyped, the genotyping error was 10.7%. 56 of the 65 muscle-colon feces sets had both sample types from the same individual successfully genotyped, giving a mean genotyping error estimate of 6.8% for fecal samples. Of the 322 retained autosomal loci, 2 had >50% missing data across individuals. Removal of these loci in future GT-seq runs may increase genotyping success, particularly for field-collected feces.
We had a total of 293 bear samples for which hunters provided a sex identity. Of these, 78.6% were genotyped successfully at one or both sex-linked loci. There were 18 instances where a sample had only a genotype at one of the sex-linked loci, and only one case of allelic dropout. For samples with genotypes at both sex loci, genotype concordance was 99.7%. Hunter identification of bear sex matched the sex genotype provided by the first sex locus for 95.9% of samples, matched the second sex locus for 91.1% of samples, and matched both sex loci for 90.1% of samples.
Table 1. GT-seq genotyping success for five different sources of polar bear DNA, including 365 total samples collected across 10 subpopulations. Shown are percent individuals successfully genotyped before and after samples with no detectable polar bear DNA were removed (based on qPCR results), and average percent missing data by locus for each sample type (individuals with >50% missing data removed).