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

Kristen M Hayward; Michelle P Harwood; Stephen C Lougheed; Zhengxin Sun; Peter Van Coeverden de Groot; Evelyn L Jensen

doi:10.7717/peerj.8884

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

PeerJ. 2020 Apr 7:8:e8884. doi: 10.7717/peerj.8884. eCollection 2020.

Authors

Kristen M Hayward^#¹, Michelle P Harwood^#^{1

2}, Stephen C Lougheed¹, Zhengxin Sun¹, Peter Van Coeverden de Groot¹, Evelyn L Jensen^{1

3}

Affiliations

¹ Department of Biology, Queen's University, Kingston, ON, Canada.
² Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
³ Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, USA.

^# Contributed equally.

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.

Keywords: Noninvasive sampling; Population genetics; Ursus maritimus; qPCR.

Grants and funding

This work was funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-123). Evelyn L. Jensen was supported by a Natural Science and Engineering Council of Canada (NSERC) postdoctoral fellowship. Michelle P. Harwood and Kristen M. Hayward were supported by Undergraduate Student Research Awards from NSERC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.