Effect of sample age and season of collection on the reliability of microsatellite genotyping of faecal DNA

Abstract

Individual identification of animals from DNA in field-collected faecal samples is becoming an increasingly important tool in wildlife population monitoring. A major issue relevant to the application of this technique is the reliability of the genotypes obtained. I investigated the effect of sample age and season of collection on amplification rates and reliability of microsatellite genotypes amplified from faecal DNA of a marsupial herbivore, the brush-tailed rock-wallaby (Petrogale penicillata) and a eutherian carnivore, the red fox (Vulpes vulpes). Comparison of DNA profiles from 1 day to 6 months for both species suggests that as the age of the faeces increases there is less good-quality DNA present on the surface of the faeces, resulting in significantly decreasing amplification rates and increasing genotyping error rates over time. No microsatellite PCR products were obtained from samples older than 3 months from any faecal DNA extract in either season. For both species, faeces collected during the summer trial yielded high-quality DNA for up to one week. Faeces collected in winter had significantly lower amplification rates and higher genotyping errors than the summer-collected samples. Computer simulations were used to estimate the probability of obtaining false genotypes when genotyping faecal samples of various ages. These revealed that three replicates is sufficient to prevent identification of false individuals for P. penicillata from faeces up to one week old in both summer and winter but more replicates may be required for older samples, particularly in winter. In contrast, up to eight replicates may be required for fox faeces collected in winter, particularly if more than one week old. These results also suggest that it is difficult to visually identify faecal age for V. vulpes, and any study using fox faeces would need to account for the likely inclusion of older faeces in a field collection. For P. penicillata, faecal age could be accurately assessed, particularly when less than one week old and targeting faeces that match the two most reliable appearance classes described here would be an efficient sampling strategy. It is recommended that the appropriate PCR replication protocol for any given study should be tailored to the error rates expected for the oldest samples likely to be collected. This study is the first to thoroughly investigate the effects of sample age and season of collection on microsatellite genotyping from faecal samples and provides guidelines for sampling and PCR repetition strategies for field-based non-invasive DNA studies.

Sex Determination of Sika Deer (Cervus nippon yesoensis) Using Nested PCR from Feces Collected in the Field

“…In such a case, several points should be kept in mind. As for factors related to the DNA condition, climate including temperature, humidity and rainfall, as well as diet or elapsed time from defecation are suggested [3,17,24]. For feces in a low DNA condition, repeating fecal DNA extraction or PCR amplification will be needed due to allelic dropout and false alleles resulting from DNA degradation [2,11,25].…”

Comparison of DNA extraction methods for polymerase chain reaction amplification of guanaco (Lama guanicoe) fecal DNA samples

“…In particular, climatic conditions and air moisture may play an important role in DNA preservation. For example, Brinkman et al (2010) reported high degradation of fecal DNA that was exposed to rainfall while Piggott (2005) obtained higher DNA quality from feces samples that were collected during summer. Because of the dry climatic conditions of our study region (Di Castri and Hajek, 1976), DNA degradation in L. guanicoe feces might be relatively slow and facilitate the amplification of small PCR products from non-fresh fecal material.…”

Noninvasive alternatives for DNA collection from threatened rodents

“…Fecal samples were collected opportunistically during each trap event. Because DNA quality is affected by fecal sample age [30,41], we ensured collection of fresh fecal samples by only using feces collected in the mesh handling bag rather than the trap. We collected all intact pellets with forceps and transferred whole pellets directly to 95% ethanol for storage; broken pellets were avoided and care was taken to avoid crushing the pellets during collection and transfer [42].…”