TECHNICAL ADVANCES: Effects of genotyping protocols on success and errors in identifying individual river otters (Lontra canadensis) from their faeces

Abstract

In noninvasive genetic sampling, when genotyping error rates are high and recapture rates are low, misidentification of individuals can lead to overestimation of population size. Thus, estimating genotyping errors is imperative. Nonetheless, conducting multiple polymerase chain reactions (PCRs) at multiple loci is time-consuming and costly. To address the controversy regarding the minimum number of PCRs required for obtaining a consensus genotype, we compared consumer-style the performance of two genotyping protocols (multiple-tubes and 'comparative method') in respect to genotyping success and error rates. Our results from 48 faecal samples of river otters (Lontra canadensis) collected in Wyoming in 2003, and from blood samples of five captive river otters amplified with four different primers, suggest that use of the comparative genotyping protocol can minimize the number of PCRs per locus. For all but five samples at one locus, the same consensus genotypes were reached with fewer PCRs and with reduced error rates with this protocol compared to the multiple-tubes method. This finding is reassuring because genotyping errors can occur at relatively high rates even in tissues such as blood and hair. In addition, we found that loci that amplify readily and yield consensus genotypes, may still exhibit high error rates (7-32%) and that amplification with different primers resulted in different types and rates of error. Thus, assigning a genotype based on a single PCR for several loci could result in misidentification of individuals. We recommend that programs designed to statistically assign consensus genotypes should be modified to allow the different treatment of heterozygotes and homozygotes intrinsic to the comparative method.

Non-invasive conservation genetics of the critically endangered golden-crowned sifaka (Propithecus tattersalli): high diversity and significant genetic differentiation over a small range

“…For all the tested samples, the same consensus genotypes were reached with fewer PCRs (22% less) than the multi-tubes approach of Taberlet et al (1996). Theses results are consistent with those obtained by Hansen et al (2008) who compared both methods using otter's faeces. These authors showed that, on average, 4.5 (±0.5) positive PCRs were needed with the comparative approach compared to an average of 6.8 (±0.6) for the multi-tubes protocols (corresponding thus to a decrease of *33%).…”

“…Our error rates (overall 5-39% for the dropout allele) were comparable to those of other studies using faeces (Morin et al 2001;Hansen et al 2008). Some loci provided much lower error rates than others (e.g., 93HDZT164 compared to 93HDZT265), and a wider-scale analysis would be required to determine the factors responsible for these differences (Taberlet et al 1999;Hansen et al 2008). Altogether our results suggest that it should be possible to significantly reduce the cost of faecal DNA studies without reducing the quality of the genotypes.…”

“…These authors showed that, on average, 4.5 (±0.5) positive PCRs were needed with the comparative approach compared to an average of 6.8 (±0.6) for the multi-tubes protocols (corresponding thus to a decrease of *33%). Our error rates (overall 5-39% for the dropout allele) were comparable to those of other studies using faeces (Morin et al 2001;Hansen et al 2008). Some loci provided much lower error rates than others (e.g., 93HDZT164 compared to 93HDZT265), and a wider-scale analysis would be required to determine the factors responsible for these differences (Taberlet et al 1999;Hansen et al 2008).…”

Comparative woodland caribou population surveys in Slate Islands Provincial Park, Ontario

“…The Mh estimator is a model derived to look at individual differences in capture probability and has relatively widespread use (Chao & Huggins, 2005). The Mh model is ideal with non-invasive genetic sampling where variability in sampling frequency for identified individuals is often apparent (Mills et al, 2000;Frantz et al, 2003;Hansen et al, 2008) and has been used previously in the estimation of population size for woodland caribou populations (Hettinga, 2010).…”

Field surveys of capercaillie (Tetrao urogallus) in the Swiss Alps underestimated local abundance of the species as revealed by genetic analyses of non-invasive samples

“…Thus, false genotypes may occur which are recorded as new capture events instead of recaptures. Methodological improvements have been proposed that reduce the risk of genotyping errors, such as scoring alleles based on several PCR replicates (Navidi et al 1992;Taberlet et al 1996;Goossens et al 2000;Hansen et al 2008). …”