Improving cost-efficiency of faecal genotyping: New tools for elephant species

Abstract

Despite the critical need for non-invasive tools to improve monitoring of wildlife populations, especially for endangered and elusive species, faecal genetic sampling has not been adopted as regular practice, largely because of the associated technical challenges and cost. Substantial work needs to be undertaken to refine sample collection and preparation methods in order to improve sample set quality and provide cost-efficient tools that can effectively support wildlife management. In this study, we collected an extensive set of forest elephant (Loxodonta cyclotis) faecal samples throughout Gabon, Central Africa, and prepared them for genotyping using 107 single-nucleotide polymorphism assays. We developed a new quantitative polymerase chain reaction (PCR) assay targeting a 130-bp nuclear DNA fragment and demonstrated its suitability for degraded samples in all three elephant species. Using this assay to compare the efficacy of two sampling methods for faecal DNA recovery, we found that sampling the whole surface of a dung pile with a swab stored in a small tube of lysis buffer was a convenient method producing high extraction success and DNA yield. We modelled the influence of faecal quality and storage time on DNA concentration in order to provide recommendations for optimized collection and storage. The maximum storage time to ensure 75% success was two months for samples collected within 24 hours after defecation and extended to four months for samples collected within one hour. Lastly, the real-time quantitative PCR assay allowed us to predict genotyping success and pre-screen DNA samples, thus further increasing the cost-efficiency of our approach. We recommend combining the validation of an efficient sampling method, the build of in-country DNA extraction capacity for reduced storage time and the development of species-specific quantitative PCR assays in order to increase the cost-efficiency of routine non-invasive DNA analyses and expand the use of next-generation markers to non-invasive samples.

More affordable and effective noninvasive single nucleotide polymorphism genotyping using high‐throughput amplicon sequencing

“…The hair samples used in this study were collected as references from captured coyotes and could therefore not be considered truly noninvasive, although the high success rate (100%) is promising for future applications using hair‐snares. Compared to the error rates of microsatellite genotyping (0%–48%) (Broquet & Petit, 2004), previous research has found that SNP‐based methods substantially reduce genotyping error rates to 1% (Kraus et al., 2015), 0.038% (Norman & Spong, 2015) and 0.29% (Bourgeois et al., 2019) when preselecting samples based on DNA concentration. Our total error rate from scat sample replicates was 1% without any preselection based on sample quality beyond selecting randomly from the subset of scats that yielded species ID via DNA metabarcoding.…”

More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing

“…The hair samples used in this study were collected as references from captured coyotes and could therefore not be considered truly noninvasive, however, the high success rate (100%) is promising for future applications using hair-snares. Compared to the error rates of microsatellite genotyping (0-48%) (Broquet & Petit, 2004), previous research has found that SNP-based methods substantially reduce genotyping error rates to 1% (Kraus et al, 2015), 0.00038% (Norman & Spong, 2015) and 0.0029% (Bourgeois et al, 2019) when pre-selecting samples based on DNA concentration. Our total error rate for scats was 0.01% without any pre-selection based on sample quality beyond selecting randomly from the subset of scats that yielded species ID via DNA metabarcoding (85% of scats yielded a species ID).…”

Factors influencing genotyping success and genotyping error rate of Eurasian otter (Lutra lutra) faeces collected in temperate Central Europe

“…Most studies on genotyping animal scats collected in the field highlighted the importance of using fresh scats to increase DNA quality (e.g. Murphy et al 2007;Vynne et al 2012;Lerone et al 2014;Bourgeois et al 2019). In the present study, we have already considered this factor and collected only otter faeces from the previous night.…”

“…Hájková et al 2006), storage duration until extraction of DNA (e.g. Soto-Calderón et al 2009;Bourgeois et al 2019) and diet of the defecating animal (Murphy et al 2003;Panasci et al 2011). Broad consensus was found regarding the age of the scat samples: genotyping success rates and sample quality were highest using the freshest samples (Piggott 2004;Murphy et al 2007;Brinkman et al 2010;Vynne et al 2012;Bourgeois et al 2019).…”

“…Soto-Calderón et al 2009;Bourgeois et al 2019) and diet of the defecating animal (Murphy et al 2003;Panasci et al 2011). Broad consensus was found regarding the age of the scat samples: genotyping success rates and sample quality were highest using the freshest samples (Piggott 2004;Murphy et al 2007;Brinkman et al 2010;Vynne et al 2012;Bourgeois et al 2019). However, since texture of faeces, diet and environmental conditions may differ between species and populations, influencing factors can vary with different animal species and even within one species occurring in different habitats (Panasci et al 2011).…”