A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts

Abstract

DNA extracted from fecal samples contains DNA from the focal species, food, bacteria and pathogens. Most DNA quantification methods measure total DNA and cannot differentiate among sources. Despite the desirability of noninvasive fecal sampling for studying wildlife populations, low amounts of focal species DNA make it difficult to use for next-generation sequencing (NGS), where accurate DNA quantification is critical for normalization. Two factors are required prior to using fecal samples in NGS libraries: (1) an accurate quantification method for the amount of target DNA and (2) a determination of the relative amount of target DNA needed for successful single nucleotide polymorphism genotyping assays. Here, we address these needs by developing primers to amplify a 101 bp region of the nuclear F2 gene and a quantitative PCR (qPCR) assay that allows the accurate quantification of the amount of polar bear (Ursus maritimus) DNA in fecal extracts. We test the assay on pure polar bear DNA extracted from muscle tissue and find a high correlation between fluorometric and qPCR quantifications. The qPCR assay was also successfully used to quantify the amount of DNA derived from polar bears in fecal extractions. Orthologs of the F2 gene have been identified across vertebrates; thus, similar qPCR assays could be developed for other species to enable noninvasive studies.

Genotyping‐in‐thousands by sequencing (GT‐seq) of noninvasive faecal and degraded samples: A new panel to enable ongoing monitoring of Canadian polar bear populations

“…Once extracted, DNA extracts from all tissue samples were run on 1.5% agarose gel stained with RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology) to assess quality and quantified using a Nanodrop ND_1000 spectrophotometer (Nanodrop Technologies Inc.). We used a polar bear-specific qPCR assay targeting the F2 gene to quantify the amount of polar bear DNA in both CF and FF samples (Hayward et al, 2020). To gauge the value of running faecal samples through this qPCR assay as a screening tool, we devised a small double-blind experiment in which we randomly divided FF samples into two subsets: 1.…”

Whole genome sequences from non-invasively collected caribou faecal samples

“…We only selected samples with no missing microsatellite genotypes, which includes ~ 82% of the samples we have genotyped since 2014. However, a better sample selection method (or to use in combination) might be to use qPCR to screen for proportion of host DNA within extractions (Snyder-Mackler et al 2016;Chiou and Bergey 2018;Hayward et al 2020), or the PCR method developed by Ball et al (2007). If used in combination with our DNA extraction technique, it is likely that genomes to the standard of our high-quality faecal genomes will be more consistently produced, further increasing the cost effectiveness of our method.…”

“…If used in combination with our DNA extraction technique, it is likely that genomes to the standard of our high-quality faecal genomes will be more consistently produced, further increasing the cost effectiveness of our method. We plan to use PCR quantification technique moving forward (Ball et al 2007;Hayward et al 2020), which should likely be standard practice for researchers choosing samples for sequencing.…”

Whole genome sequences from non-invasively collected samples

“…To select which samples to sequence, we looked at raw genotype peaks from microsatellite scores to assess quality and endogenous DNA content. However, a better method (or to use in combination) might be to use qPCR to screen for proportion of host DNA within extractions (Chiou & Bergey, 2018;Hayward et al, 2020;Snyder-Mackler et al, 2016), or the PCR method developed by Ball et al (2007). If used in combination with our DNA extraction technique, it is likely that genomes to the standard of our high quality faecal genomes will be more consistently produced, further increasing the cost effectiveness of our method.…”

“…If used in combination with our DNA extraction technique, it is likely that genomes to the standard of our high quality faecal genomes will be more consistently produced, further increasing the cost effectiveness of our method. Unfortunately, we have no more DNA left from the extractions used for our faecal genomes so we cannot screen them post-hoc to test for a correlation, but we plan to use PCR quantification technique moving forward for the next batch of faecal genomes (Ball et al, 2007;Hayward et al, 2020). This should likely be standard practice for researchers choosing samples for sequencing.…”