Noninvasive genotyping methods have become key elements of wildlife research over the last two decades, but their widespread adoption is limited by high costs, low success rates and high error rates. The information lost when genotyping success is low may lead to decreased precision in animal population densities, which could misguide conservation and management actions. Single nucleotide polymorphisms (SNPs) provide a promising alternative to traditionally used microsatellites as SNPs allow amplification of shorter DNA fragments, are less prone to genotyping errors and produce results that are easily shared among laboratories. Here, we outline a detailed protocol for cost‐effective and accurate noninvasive SNP genotyping using multiplexed amplicon sequencing optimized for degraded DNA. We validated this method for individual identification by genotyping 216 scats, 18 hairs and 15 tissues from coyotes (Canis latrans) using 26 SNPs. Our genotyping success rate for scat samples was 93%, and 100% for hair and tissue, representing a substantial increase compared to previous microsatellite‐based studies while remaining at a low cost of under $5 per PCR replicate (excluding labour). The accuracy of the genotypes was further corroborated in that genotypes from scats matching known, GPS‐collared coyotes were always located within the territory of the known individual. We also show that different levels of multiplexing produced similar results, but that PCR product cleanup strategies can have substantial effects on genotyping success. By making noninvasive genotyping more affordable, accurate and efficient, this research may allow for a substantial increase in the use of noninvasive methods to monitor and conserve free‐ranging wildlife populations.

中文翻译：

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使用高通量扩增子测序进行更实惠、更有效的无创单核苷酸多态性基因分型。

在过去的二十年里，无创基因分型方法已成为野生动物研究的关键要素，但它们的广泛采用受到高成本、低成功率和高错误率的限制。基因分型成功率低时丢失的信息可能会导致动物种群密度的精确度降低，从而误导保护和管理行动。单核苷酸多态性 (SNP) 为传统使用的微卫星提供了一种有前景的替代方案，因为 SNP 允许扩增较短的 DNA 片段，不太容易出现基因分型错误并产生易于在实验室之间共享的结果。在这里，我们概述了使用针对降解 DNA 优化的多重扩增子测序进行经济高效且准确的无创 SNP 基因分型的详细方案。犬类) 使用 26 个 SNP。我们对粪便样本的基因分型成功率为 93%，对头发和组织的基因分型成功率为 100%，与之前的基于微卫星的研究相比，显着增加，同时保持每个 PCR 复制（不包括人工）低于 5 美元的低成本。基因型的准确性得到进一步证实，因为来自与已知的、带有 GPS 项圈的土狼相匹配的粪便的基因型总是位于已知个体的领土内。我们还表明，不同水平的多路复用产生了相似的结果，但 PCR 产物净化策略可能对基因分型成功产生重大影响。通过使非侵入性基因分型更加经济、准确和高效，这项研究可能会大大增加非侵入性方法的使用，以监测和保护自由放养的野生动物种群。