Two‐step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples

Abstract

Many studies in molecular ecology rely upon the genotyping of large numbers of low-quantity DNA extracts derived from noninvasive or museum specimens. To overcome low amplification success rates and avoid genotyping errors such as allelic dropout and false alleles, multiple polymerase chain reaction (PCR) replicates for each sample are typically used. Recently, two-step multiplex procedures have been introduced which drastically increase the success rate and efficiency of genotyping. However, controversy still exists concerning the amount of replication needed for suitable control of error. Here we describe the use of a two-step multiplex PCR procedure that allows rapid genotyping using at least 19 different microsatellite loci. We applied this approach to quantified amounts of noninvasive DNAs from western chimpanzee, western gorilla, mountain gorilla and black and white colobus faecal samples, as well as to DNA from ~100-year-old gorilla teeth from museums. Analysis of over 45 000 PCRs revealed average success rates of > 90% using faecal DNAs and 74% using museum specimen DNAs. Average allelic dropout rates were substantially reduced compared to those obtained using conventional singleplex PCR protocols, and reliable genotyping using low (< 25 pg) amounts of template DNA was possible. However, four to five replicates of apparently homozygous results are needed to avoid allelic dropout when using the lowest concentration DNAs (< 50 pg/reaction), suggesting that use of protocols allowing routine acceptance of homozygous genotypes after as few as three replicates may lead to unanticipated errors when applied to low-concentration DNAs.

Cues to kinship and close relatedness during infancy in white-faced capuchin monkeys, Cebus capucinus

“…The observed heterozygosity was estimated using all genotyped individuals in the Lomas population, including those analyzed by Muniz et al (2006). Allelic dropout rates were determined by looking at those samples analyzed by IG; we limited data to heterozygous loci, calculated the proportion of times that the loci was falsely scored as homozygous, and divided those numbers over the total number of PCRs for the loci as per Arandjelovic et al (2009 Table S8: GLMM results for probability that an adult male is the father of an infant.…”

“…Genetic information for 172 capuchins from the Lomas Barbudal population was available from previously published work (Muniz et al, 2006) and we reanalyzed DNAs from nine individuals from that study to ensure consistency in allele calling. The PCR protocol ) was adapted to allow for two-step multiplex PCR (Arandjelovic et al, 2009). Males under six years of age would only be considered potential sires if we had good demographic records and, in using CERVUS we could not identify a sire with high statistical confidence.…”

Genetic differentiation and the evolution of cooperation in chimpanzees and humans

“…Genotypes from five chimpanzee groups were previously published [22,[40][41][42], while genotypes for 13 groups were newly generated for this study. We used a two-step amplification method, where all 19 loci were combined with template DNA in an initial multiplex PCR reaction, with dilutions of the resultant PCR products subsequently amplified in singleplex PCR reactions using fluorescently labelled forward primers and unlabelled Chimp and human genetic differentiation K. Langergraber et al 2547 nested reverse primers [43]. We performed the necessary number of PCR replications to produce error rates of less than 1 per cent, as established in previous work [43].…”

“…We used a two-step amplification method, where all 19 loci were combined with template DNA in an initial multiplex PCR reaction, with dilutions of the resultant PCR products subsequently amplified in singleplex PCR reactions using fluorescently labelled forward primers and unlabelled Chimp and human genetic differentiation K. Langergraber et al 2547 nested reverse primers [43]. We performed the necessary number of PCR replications to produce error rates of less than 1 per cent, as established in previous work [43]. Eleven of the chimpanzee groups were habituated or semihabituated to human observation, facilitating the collection of faecal samples from identified adult individuals.…”

A comparative study of the use of tiger-specific and heterologous microsatellite markers for population genetic studies of the Bengal tiger (Panthera tigris tigris)

“…However, at the same time, the use of a greater number of loci may introduce more genotyping errors when using non-invasively collected samples of a low-quality source of DNA (Creel et al, 2003). This problem may be minimized by using the multiple-tube approach (Navidi et al, 1992;Goossens et al, 1998) and two-step multiplex PCR method without compromising with number of loci (Arandjelovic et al, 2009;Chang et al, 2012) needed to understand species biology and ecology.…”