Age is just a number: No impacts of scat ageing on single nucleotide polymorphism genotyping using a target capture approach

Recent advances in DNA sequencing and genotyping technologies are rapidly building our capacity to address ecological, evolutionary, and conservation questions for wildlife. However, wildlife genetic research increasingly relies on samples containing low quantities and quality of DNA, such as non‐invasive, archival, and environmental DNA. These samples present unique methodological challenges; for samples collected in the wild, it is important to understand the impact of environmental exposure time or sample ‘age’ on DNA quality and downstream genetic analyses. Here, we aged koala (Phascolarctos cinereus) scats (from five wild adult individuals temporarily housed while in veterinary care) under natural conditions and quantified DNA degradation. We assessed the effect of age (0, 1, 2, 3, 5, and 10 days) on genetic data quality by measuring the proportion of missing single-nucleotide polymorphism (SNP) data using DArTCap, a targeted genotyping methodology hypothesised to tolerate degraded DNA. Contrary to other studies, we found koala scat age was not a significant predictor of genotyping quality (i.e., rate of missing SNP calls) in the first 10 days of environmental exposure. We yielded high quality data from 10‐day‐old scats, but also low‐quality data from fresh scats. This study is the first to investigate the effect of scat age on genotyping success using a targeted approach, and DArTCap specifically. These findings support the use of targeted genotyping (such as DArTCap) from scats and provide insights for future research using DNA from non‐invasive samples. Targeted genotyping may extend the timeframe, from which accurate data can be obtained from non‐invasive samples, increasing potential sample sizes and enhancing our ability to address important questions in population ecology, conservation genetics, and population management.

Methods
Fresh scats were collected from five temporarily housed wild adult koalas (two females, three males) that were generally in good health from areas around Southeast Queensland. Scats were collected from the ground of each individual koala's enclosure in the morning; all enclosures had been cleaned the night before. Six pellets were collected from each individual. 

One scat from each koala was frozen immediately (−20°C). Based on the time at which koala cages were cleaned, these scats were less than 12 h old (named 0D in results). To simulate the effect of natural environmental degradation processes on scats over time (scat age), the remaining five scats from each koala were placed on individual toothpicks embedded into five Styrofoam boards. To simulate natural tree and ground cover conditions, Styrofoam boards with scats were then placed within a mesh enclosure and located near the base of a tree in a remnant forest patch. 

Scats were sampled for DNA extraction at six time points: 0 (< 12 h old), 1, 2, 3, 5, and 10 days (N = 30 scats, six scats per five individual koalas). At each time point, one scat per individual was removed from the outdoor setup and transferred to the laboratory and frozen at −20°C. The exfoliated koala intestinal epithelial cells present on the surface of the scat were targeted by slicing off and retaining the outer‐most layer of the scat using a sterile scalpel. DNA was then extracted from these outer surface slices using the QIAmp PowerFecal Pro DNA kit (Qiagen).

One DNA sample for each individual from each sampling time point was used for SNP genotyping. SNP genotyping was conducted by Diversity Arrays Technology, Canberra, using DArTCap technology. DArTCap involves a combination of complexity reduction methods and next‐generation sequencing platforms.