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).