Frank Lexi E, Lindsey Laramie L, Kipp Evan J, Faulk Christopher, Stone Suzanne, Roerick Tanya M, Moore Seth A, Wolf Tiffany M, Larsen Peter A
Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, MN 55108, United States.
Department of Animal Science, University of Minnesota, St. Paul, MN 55108, United States.
J Mammal. 2024 May 6;105(5):965-975. doi: 10.1093/jmammal/gyae044. eCollection 2024 Oct.
Accurate taxonomic species identification is essential to the study of mammals. Despite this necessity, rapid and accurate identification of cryptic, understudied, and elusive mammals remains challenging. Traditional barcoding of mitochondrial genes is standard for molecular identification but requires time-consuming wet-lab methodologies. Recent bioinformatic advancements for nanopore sequencing data offer exciting opportunities for noninvasive and field-based identification of mammals. Nanopore adaptive sampling (NAS), a polymerase chain reaction (PCR)-free method, selectively sequences regions of DNA according to user-specified reference databases. Here, we utilized NAS to enrich mammalian mitochondrial genome sequencing to identify species. Fecal DNA extractions were sequenced from 9 mammals, several collected in collaboration with Minnesota Tribal Nations, to demonstrate utility for NAS barcoding of noninvasive samples. By mapping to the entire National Center for Biotechnology Information mammalian mitochondrial reference genome database and bioinformatically analyzing highly similar matches, we successfully produced species identifications for all fecal samples. Eight of 9 species identifications matched previous PCR or animal/fecal appearance-based identifications. For the ninth species, our genetic data indicate a misidentification stemming from the original study. Our approach has a range of applications-particularly in field-based wildlife research, conservation, disease surveillance, and monitoring of wildlife trade. Of importance to Minnesota tribes is invasive species monitoring, detections, and confirmation as climate impacts cause changes in biodiversity and shifts in species distributions. The rapid assessment techniques described here will be useful as new introductions and range expansions of native and invasive species may first be detected by the presence of signs such as scat rather than direct observations and will be helpful for chronically understaffed tribal natural resources agencies.
准确的分类物种鉴定对于哺乳动物的研究至关重要。尽管有此必要性，但对隐秘、研究不足且难以捉摸的哺乳动物进行快速准确的鉴定仍然具有挑战性。传统的线粒体基因条形码技术是分子鉴定的标准方法，但需要耗时的湿实验室方法。最近纳米孔测序数据的生物信息学进展为哺乳动物的非侵入性和基于野外的鉴定提供了令人兴奋的机会。纳米孔自适应采样（NAS）是一种无需聚合酶链反应（PCR）的方法，可根据用户指定的参考数据库选择性地对DNA区域进行测序。在这里，我们利用NAS来富集哺乳动物线粒体基因组测序以鉴定物种。对9种哺乳动物的粪便DNA提取物进行了测序，其中几种是与明尼苏达部落国家合作采集的，以证明NAS对非侵入性样本进行条形码鉴定的效用。通过映射到整个美国国立生物技术信息中心哺乳动物线粒体参考基因组数据库并对高度相似的匹配进行生物信息学分析，我们成功地对所有粪便样本进行了物种鉴定。9个物种鉴定中有8个与之前基于PCR或动物/粪便外观的鉴定相匹配。对于第九个物种，我们的遗传数据表明最初的研究存在错误鉴定。我们的方法有一系列应用——特别是在基于野外的野生动物研究、保护、疾病监测以及野生动物贸易监测方面。对明尼苏达部落来说重要的是入侵物种的监测、检测和确认，因为气候影响导致生物多样性变化和物种分布转移。这里描述的快速评估技术将很有用，因为本地和入侵物种的新引入和范围扩展可能首先通过粪便等迹象的出现而被检测到，而不是直接观察，这将有助于长期人员不足的部落自然资源机构。
