Droplet Digital PCR Provides Highly Sensitive and Accurate Opsin Gene SNP Detection From Wild Primate Fecal Samples

Area of Science:

• Molecular Biology
• Genetics
• Primate Ecology

Background:

• Evaluating low-quality, low-quantity DNA from field samples like feces is challenging for molecular biology.
• Innovations in Polymerase Chain Reaction (PCR) technology show promise in overcoming these limitations.
• Color vision in New World monkeys is linked to opsin gene single nucleotide polymorphisms (SNPs).

Purpose of the Study:

• To evaluate the efficiency of droplet digital PCR (ddPCR) for noninvasive color vision assessment in white-faced capuchins (Cebus imitator).
• To determine allele and genotype frequencies for color vision-related opsin gene SNPs in a wild capuchin population.
• To compare the sensitivity and reliability of ddPCR versus Sanger sequencing for genotyping from fecal DNA.

Main Methods:

• DNA extraction from fecal samples of 211 wild capuchins.
• Genotyping using droplet digital PCR (ddPCR) and Sanger sequencing.
• Comparison of ddPCR and Sanger sequencing for accuracy and sensitivity on the same opsin gene targets.

Main Results:

• Droplet digital PCR (ddPCR) demonstrated higher sensitivity and required fewer samples than Sanger sequencing for definitive genotyping.
• Both methods yielded identical opsin genotypes, confirming ddPCR's reliability.
• The most frequent phenotypes observed were red and green/red; genotype distributions varied between sexes.

Conclusions:

• Droplet digital PCR (ddPCR) is a reliable, highly sensitive, and high-throughput method for noninvasive color vision assessment in wild capuchins using fecal samples.
• ddPCR's robustness to PCR inhibitors makes it suitable for analyzing challenging field samples.
• This technique has potential applications for noninvasively identifying other disease-related SNP mutations in wild animal populations.