Allelic Dropout Rates
Here we recover high rates of detected allelic dropout in samples
classified as low quality museum specimens from many rounds of PCR
amplification of nuclear and mitochondrial DNA markers. The rates of
allelic dropout averaged 12.6% across the five evaluated microsatellite
loci for the pooled dataset. When we applied our ‘best practices’ method
from this study the average rate of allelic dropout was reduced to 9.8%
across all loci and including the LQMS. When data from the pooled run
was evaluated, our rates of allelic dropout in the LQMS (average of
19.6%) conformed to rates reported for various studies of avian fecal
allelic dropout (mean of 21%, Regnaut, Lucas, & Fumagalli, 2006) but
was much higher than rates reported from chimpanzee fecal samples via
GBS (7% rate of allelic dropout; Barbian et al., 2018). This may be due
to the fact that Barbian et al., (2018) only genotyped samples at loci
that had over 500 reads (counts.min = 500). After manually processing
genotypes from all the a priori information ( the replicates, pooled
run, and bioinformatically pooled data) the rates of dropout in the LQMS
increased dramatically following processing to 79%. However, that was
due to most genotypes lacking verification, and resulted in only four
total genotypes called across all LQMS samples, with MVZ 2088 recovering
three genotypes and MVZ 5211 recovering one. It is also worth mentioning
that the LQMS samples had very high rates of individual allelic dropout
due to unreliable genotypes which were ultimately removed in the
manually processed (best practices) genotypes.
The HQMS samples performed better than expected, all recovering very low
rates of allelic dropout (2.4% from the pooled run and
<0.001% following our best practices). Two samples were
collected from 1926 and one from 1975, and provide robust evidence for
the utility of museum specimens for the recovery of microsatellite
genotypes. These three samples performed on par with GBS studies derived
from tissue samples (Darby et al., 2016).
The rate of allelic dropout in our tissue sample was higher upon raw
results from CHIIMP, at 14.7% compared to 0.4% in Darby et al.,
(2016). However, this was partially due to one instance of primer region
trimming (GS-4) for this sample. Following implementation of our best
practices the rate was reduced to 8.5%, which is still higher than
other studies of tissue samples (Darby et al., 2016). Additionally, the
aberrant call of 251 from HSU 8180 (GSLA-52, pooled run) was removed
during our best practices.
The pooled run CHIIMP results for dropout when separated by length were:
7.5% for short, 0.35% for medium and 48% for long loci. The higher
rates for the short loci is likely attributed to non-specific
amplification or higher amounts of stutter and PCR artifacts as
identified from CHIIMP. Following our manually processed genotypes we
recovered 0.0025%, 6.3%, and 36% occurrence of allelic dropout for
short, medium and long loci.