Hervey Samuel D, vonHoldt Bridgett M, Romanski Mark C, Wheeldon Tyler J, Patterson Brent R, Brzeski Kristin E
College of Forest Resources and Environmental Sciences Michigan Technological University Houghton Michigan USA.
Department of Ecology and Evolutionary Biology Princeton University Princeton New Jersey USA.
Ecol Evol. 2025 Apr 21;15(4):e71240. doi: 10.1002/ece3.71240. eCollection 2025 Apr.
The application of noninvasive genetic methods toward the field of conservation has increased our understanding of many wildlife populations that are difficult to sample, allowing for better management. In molecular ecology, the use of noninvasive sampling became widely feasible with the advent of microsatellites, a highly polymorphic, short-length marker that could be genotyped from low-quality DNA sources. Despite decades of use, many microsatellite panels continue to suffer from high genotyping error rates, allelic dropout, and limited reproducibility across laboratories. To address these issues, single nucleotide polymorphisms (SNPs) offer advantages such as lower genotyping error rates, avoidance of allelic dropout due to consistent allele length, and automated calling through bioinformatic pipelines, reducing human subjectivity and error. Given the advantages SNPs provide relative to microsatellites as a molecular marker, the use of SNP panels and specifically, the method of genotyping-in-thousands by sequencing (GTseq) has gained popularity. Here, we developed a GTseq panel for western Great Lakes canids comprised of 196 loci, capable of species identification, accurately inferring sex (97.2%), identifying unique individuals (probability of identity = 6.71e), assigning relationships (false positive rate = 9.34e), and assigning genotypes with low error (0.39%). In an attempt to improve genotyping success with low-quality samples, we found that while increasing the number of PCR cycles yielded a higher percentage of genotyped loci, it also increased on-target reads in negative PCR controls. We suggest approaching this manipulation with caution and emphasize the importance of including and reporting negative PCR controls. Further, quantitative PCR was a powerful method to estimate host-specific DNA concentrations, enabling conservative sample selection for library preparation with respect to GTseq affordability.
非侵入性遗传方法在保护领域的应用增进了我们对许多难以采样的野生动物种群的了解，有助于更好地进行管理。在分子生态学中，随着微卫星的出现，非侵入性采样变得广泛可行，微卫星是一种高度多态的短长度标记，可以从低质量DNA来源进行基因分型。尽管使用了数十年，但许多微卫星面板仍然存在基因分型错误率高、等位基因缺失以及不同实验室间再现性有限的问题。为了解决这些问题，单核苷酸多态性（SNP）具有一些优势，如较低的基因分型错误率、由于等位基因长度一致而避免等位基因缺失，以及通过生物信息学管道进行自动判型，减少了人为主观性和误差。鉴于SNP相对于微卫星作为分子标记所具有的优势，SNP面板的使用，特别是通过测序进行数千样本基因分型（GTseq）的方法越来越受欢迎。在此，我们为大湖西部地区的犬科动物开发了一个由196个位点组成的GTseq面板，该面板能够进行物种鉴定、准确推断性别（97.2%）、识别独特个体（个体识别概率 = 6.71e）、确定亲缘关系（假阳性率 = 9.34e）以及以低错误率（0.39%）确定基因型。为了提高低质量样本的基因分型成功率，我们发现虽然增加PCR循环次数会使基因分型位点的百分比更高，但也会增加阴性PCR对照中的靶向读数。我们建议谨慎进行这种操作，并强调纳入和报告阴性PCR对照的重要性。此外，定量PCR是估计宿主特异性DNA浓度的有效方法，对于GTseq的成本效益而言，能够在文库制备时进行保守的样本选择。
