Evaluation of fecal DNA preservation techniques and effects of sample age and diet on genotyping success

Abstract

Optimal collection and preservation protocols for fecal DNA genotyping are not firmly established. We evaluated 3 factors that influence microsatellite genotyping success of fecal DNA extracted from coyote (Canis latrans) scats: 1) age of scat, 2) preservative, and 3) diet content. We quantified genotyping success by comparing rates of allelic dropout, false alleles, and failed amplifications among consensus genotypes. We used a panel of 6 microsatellite loci to genotype 20 scat samples, each of which was subjected to 3 age (1 day, 5 days, and 10 days post‐deposition) and 3 preservation (DET buffer, 95% ethanol [EtOH], and lysis buffer) treatments. Both sample age and storage buffer had a significant effect on success and reliability. Ethanol and DET buffer preserved fecal samples with similar efficiency, and both were superior to lysis buffer. Our analysis of DNA degradation rates revealed that samples collected as early as 5 days of age yielded DNA that was highly degraded relative to samples collected on day 1. We tested the influence of dietary remains on microsatellite genotyping by using scat samples consisting predominantly of insect prey (n = 5), mammalian prey (n = 9), or the remains of juniper (Juniperus spp.) berries (n = 6) and compared EtOH and DET buffer preservation efficacy. We observed a significant interaction effect between storage buffer and diet for the probability of a false allele in a polymerase chain reaction (PCR), suggesting that the optimal preservation technique depended on the food remains comprising the scat. Scats comprised of juniper berry remains were more reliably genotyped when preserved in DET than EtOH. Mammalian prey‐based scats were more reliable when stored in EtOH than DET buffer. Insect‐predominant scats were preserved in EtOH and DET buffer with similar efficiency. Although accurate and reliable results can be obtained from scats collected at ≥5 days of age, we suggest sampling design to include collection of scats <5 days of age to minimize field and laboratory expenses. We suggest EtOH preservation for scats of obligate carnivores and of facultative carnivores with a diet consisting primarily of mammals. We suggest DET buffer preservation for animals with a diet consisting of plant‐derived foods. Lysis buffer protocols that we employed should not be used for fecal DNA preservation. © 2011 The Wildlife Society.

Evaluating preservation medium for the storage of DNA in African lion Panthera leo faecal samples

“…Panasci et al (2011) showed that coyote faeces preserved in 95% ethanol and DETs buffer performed equally well and both performed better than samples stored in lysis buffer. Wasser et al (1997) and Panasci et al (2011) advised against the use of lysis buffer for the preservation of faecal DNA because it could result in the digestion of ingested hair from consumed prey that thereby could act as a potential source of DNA contamination. Also, it is possible that faeces collected in ASL buffer get lysed if stored at room temperature prior to deposition in the freezer.…”

“…These studies span an array of mammal species (Appendix 1) including several species of carnivores (e.g. Wasser et al, 1997;Murphy et al, 2002;Santini et al, 2007;Panasci et al, 2011;Reddy et al, 2012). A few studies comparing storage methods in tropical environments have been conducted mostly on primates (Gerloff et al, 1995;Frantzen et al, 1998;Bayes et al, 2000;Nsubuga et al, 2004;Vallet et al, 2007).…”

“…Several studies have evaluated the relative genotyping success from two or more preservation media (Wasser et al, 1997;Frantzen et al, 1998;Murphy et al, 2002;Panasci et al, 2011). These studies span an array of mammal species (Appendix 1) including several species of carnivores (e.g.…”

OPTIMIZACIÓN DE UNA TÉCNICA PARA LA EXTRACCIÓN DE UN ADN DE HECES DE VICUÑA (Vicugna vicugna mensalis).

“…El análisis genético a partir de ADN extraído de heces es complicado y engorroso, pues generalmente el ADN obtenido se encuentra contaminado con restos propios del material fecal, de la dieta del animal o suele estar en bajas cantidades o degradado (Deuter et al, 1995; Kohn et al, 1995;Mak et al, 2004;Panasci et al, 2011). El uso de un método adecuado de extracción es fundamental, no solamente para eliminar impurezas, pero sobre todo, para evitar la inhibición en la amplificación de productos en pruebas de PCR.…”

Aportes de la genética de la conservación al estudio de los mamíferos neotropicales: revisión y análisis crítico

“…Particularmente, para los mamíferos se han realizado distintos esfuerzos por optimizar la obtención de ADN a partir de pelos y heces. Siguiendo esta línea, se pusieron a punto trampas de pelo para distintas especies (Castro-Arellano et al 2008;Biondo et al 2010;García-Alaniz et al 2010) y se propusieron protocolos para aumentar la eficiencia de las extracciones de ADN a partir de heces mediante la remoción de los inhibidores que éstas contienen (Hebert et al 2011;Nardelli et al 2011;Panasci et al 2011;Maturrano et al 2012). Por otro lado, Gomes Rocha et al (2015) lograron recientemente recuperar ADN de pequeños mamíferos a partir de huesos presentes en egagrópilas de la lechuza común, Tyto alba.…”