Effects of time and environmental conditions on the quality of DNA extracted from fecal samples for genotyping of wild deer in a warm temperate broad-leaved forest

Abstract

Extraction of DNA from non-invasive samples (feces) has been used increasingly in genetic research on wildlife. For effective and reliable genetic analyses, knowledge about which samples should be selected in the field is essential. For this reason, we examined the process of DNA degradation in feces of deer. We collected fresh fecal pellets from three wild deer living in a warm temperate forest.We then assessed the effects of time (3, 5, and 10 days) under three environmental conditions (on the forest floor, on exposed ground, and inside the laboratory) on the rates of correct genotyping (CG), amplification failure (NA), genotyping error among positive amplification (ER), false alleles (FA), and allelic dropout (AD) of 15 microsatellite loci. The rate of CG significantly decreased, and those of NA and FA increased with increasing lapse of time. Rates of CG tended to be highest and those of NA, ER, FA, and AD to be lowest in feces kept inside, followed by those on the forest floor. Suitability of samples for DNA extraction was lowest in fecal pellets left on exposed ground, and we suspect that rain may hasten DNA degradation. NA rate could serve as a reliable indicator of the quality of fecal pellets because it was significantly positively correlated with ER rate. For efficient genetic analyses using deer feces in warm temperate zones, we recommend collecting fecal pellets within 3 days of defecation, during periods without rainfall and from under the cover of trees.

A DNA toolbox for non-invasive genetic studies of sambar deer (Rusa unicolor)

“…Decreased DNA quantity and DNA degradation is also exacerbated by weathering, especially rainfall (Agetsuma Yanagihara et al 2017). Consequently, the collection of scats for genetic studies should be conducted in dry conditions and during fine weather.…”

“…Despite the many benefits of non-invasive sampling, the use of DNA sourced from discarded biological samples such as scats is associated with some difficulties. These are related to the rapid degradation of DNA following cell death (Alaeddini et al 2010), a process which may be accelerated by environmental factors including rain, or by sample storage conditions (Agetsuma Yanagihara et al 2017;Wedrowicz et al 2013). Studies that utilise non-invasive genetic sampling are, therefore, often constrained by low DNA quantity (DNA amount) and quality (DNA integrity) which can cause errors in the genetic data obtained, leading to incorrect findings and conclusions (Bonin et al 2004).…”

Superior performance of human wastewater-associated viral markers compared to bacterial markers in tropical environments

“…However, differences in reported quantity could result from intrinsic factors, such as different gut microbial levels due to diets and lifestyles (Honap et al, 2020; Mah et al, 2008; Turnbaugh et al, 2009), and extrinsic factors, such as temperature, sewer pipe condition, and retention time (Ahmed et al, 2019c; Okadera et al, 2020). Comparisons among different studies could also present confounding biases due to different field and laboratory protocols, analytical instruments, and data analysis methods, which could affect each virus at different levels (Agetsuma-Yanagihara et al, 2017; Crank et al, 2020; Kongprajug et al, 2020; Petcharat et al, 2020; Shanks et al, 2012).…”

Towards high–throughput analyses of fecal samples from wildlife

“…There is a low amount of endogenous DNA that comes from the cells swept away by the outer layer of the feces on their transit through the digestive track (Taberlet, 1996), which is normally less than the amount of micro-organismal DNA. Finally, environmental conditions, chemical compounds, and microorganisms can fragment and degrade DNA decreasing the likelihood of amplification through PCR (Deagle et al, 2006;Brinkman et al, 2010;Panasci et al, 2011;Demay et al, 2013;Roques et al, 2014;Agetsuma-Yanagihara et al, 2017). Such processes can lead to PCR failure, allelic dropout (failure to amplify one of two alleles), and/or false alleles (Taberlet, 1996;Miller et al, 2002;Broquet & Petit, 2004;Panasci et al, 2011).…”