Assessing the levels of microsatellite allelic dropout in museum
specimens using high-throughput sequencing and genotyping by synthesis.
The use of museum specimens held in natural history repositories for
population and conservation genetic research is increasing in tandem
with the use of next generation sequencing technologies. Short Tandem
Repeats (STRs), or microsatellite loci, are commonly used genetic
markers in population and conservation genetic studies. However, they
traditionally suffered from a host of issues: fragment size homology,
high costs, and low throughput as a result of capillary electrophoresis
genotyping and difficulty in reproducibility across laboratories. Next
generation sequencing technologies can address these problems, but the
incorporation of DNA derived from museum specimens suffers from
significant fragmentation and contamination with exogenous DNA.
Combatting these issues requires extra measures of stringency in the lab
and during data analysis, yet there have not been any studies evaluating
microsatellite allelic dropout from museum specimen extracted DNA. In
this study, we explore a high throughput sequencing method to evaluate
the amount of variation found within museum specimen DNA extracts for
previously characterized microsatellites across PCR replicates. We found
it useful to classify samples based on quality after replicated PCRs,
which determined the rate by which genotypes were accurately recovered.
We also found that longer microsatellites performed worse in all museum
specimens, so when designing a study invoking museum specimens, short
markers (under 250 bp) should be preferentially selected. Allelic
dropout rates across loci were dependent on sample quality. The high
quality museum specimens performed as well, and recovered nearly as high
quality metrics as our tissue sample. Mitochondrial DNA sequences were
not predictive of nuclear DNA presence, as all samples recovered
cytochrome b fragments yet many lacked microsatellite genotypes,
particularly in samples deemed low quality. Based on our results, we
have provided a set of best practices for screening, quality assurance,
and incorporation of reliable genotypes from museum specimens.