Microsatellite Recovery
Microsatellite genotypes were recovered at a higher rate than expected
based on failed results from low quality samples in PCR. The LQMS
recovered an overall PCR success rate of 21.4% (calculated from the
average success for all recorded PCRs of the LQMS from Appendix 1) yet
recovered 42% genotyping success (calculated from a total of 30
recovered genotypes from the possible 72 for the LQMS, Table 4). This
percentage did not include removal of problematic genotypes. When only
genotypes that were agreeable were included, this reduced the 30
genotypes to 6 or 8.3%. Despite this very low rate of confirmation
among the LQMS this study quantifies rates for genotyping success via
GBS on poorly amplifying museum specimens for the first time.
Alternatively, for HQMS the rate of recovered genotypes was 91.7% (66
of 72), and 86.1% (62 of 72) of agreeable genotypes. For the tissue
sample 100% (16 of 16) of the replicates resulted in a genotype, with
87.5% (14 of 16) confirmed by the second genotype recovered. This
provides robust support that the rate of disagreement shown in the HQMS
is negligible and only 1.4% less than the tissue sample.
Samples which routinely amplified produced reliable genotypes. Low
quality samples had variable genotypes which seemed stochastic and
unreliable across various PCR replicates. Additionally, it appeared that
these variable genotypes may have resulted from differences between
replicated PCR. This was apparent when sequencing reads were
bioinformatically combined as well as from the pooled run. Short
microsatellites amplified more frequently and certain microsatellites
had more reliable calls than others. This study has shown that samples
which do not reliably amplify as assessed by gel electrophoresis may be
prone to poor performance in PCR and result in inaccurate genotypes.
We were not able to compare the resulting genotypes recovered here to
other population genetics studies on G. oregonensis , as this is
the first time such a study has occurred in this species. The study
performed by Barbian et al., (2018) used GBS to re-genotype chimpanzees
with known life-history data and previous capillary electrophoresis (CE)
genotypes, and noted a shift of 1-3 bp in genotype results between the
CE and GBS data. They also noted the recovery of additional allelic
diversity, which was traditionally lost in homoplasy (and confirmed by
pedigree analysis). We also recovered homoplastic events (detailed in
Appendix 5) in our data. Darby et al., (2016) noted a 44% increase in
alleles due to homoplasy in their dataset as well, and increased alleles
from 164 to 294. In other words, 130 novel alleles were recovered from
GBS over CE.