Hayward Kristen M, Harwood Michelle P, Lougheed Stephen C, Sun Zhengxin, Van Coeverden de Groot Peter, Jensen Evelyn L
Department of Biology, Queen's University, Kingston, ON, Canada.
Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
PeerJ. 2020 Apr 7;8:e8884. doi: 10.7717/peerj.8884. eCollection 2020.
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 gene and a quantitative PCR (qPCR) assay that allows the accurate quantification of the amount of polar bear () 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 gene have been identified across vertebrates; thus, similar qPCR assays could be developed for other species to enable noninvasive studies.
从粪便样本中提取的DNA包含目标物种、食物、细菌和病原体的DNA。大多数DNA定量方法测量的是总DNA，无法区分其来源。尽管非侵入性粪便采样对于研究野生动物种群很有吸引力，但目标物种的DNA含量低，难以用于下一代测序（NGS），而在NGS中准确的DNA定量对于标准化至关重要。在将粪便样本用于NGS文库之前，需要两个因素：（1）一种准确的目标DNA量定量方法，以及（2）确定成功进行单核苷酸多态性基因分型测定所需的目标DNA相对量。在这里，我们通过开发引物来扩增核基因的一个101 bp区域以及一种定量PCR（qPCR）测定法来满足这些需求，该测定法能够准确量化粪便提取物中北极熊（）DNA的量。我们在从肌肉组织中提取的纯北极熊DNA上测试了该测定法，发现荧光定量和qPCR定量之间具有高度相关性。qPCR测定法也成功用于量化粪便提取物中源自北极熊的DNA量。该基因的直系同源基因已在脊椎动物中得到鉴定；因此，可以为其他物种开发类似的qPCR测定法，以实现非侵入性研究。
