Raw sequencing data and ngsfilters for snow track eDNA samples

Continued advancements in environmental DNA (eDNA) research have made it possible to access intraspecific variation from eDNA samples, opening new opportunities to expand non-invasive genetic studies of wildlife populations. However, the use of eDNA samples for individual genotyping, as typically performed in non-invasive genetics, still remains elusive. We present the first successful individual genotyping of eDNA obtained from snow tracks of three large carnivores: brown bear (Ursus arctos), European lynx (Lynx lynx) and wolf (Canis lupus). DNA was extracted using a protocol for isolating water eDNA and genotyped using amplicon sequencing of short tandem repeats (STR) and, for brown bear, a sex marker, on a high-throughput sequencing platform. Individual genotypes were obtained for all species, but genotyping performance differed among samples and species. The proportion of samples genotyped to individuals was higher for brown bear samples (5/7) than for wolf (7/10) and lynx (4/9), but locus genotyping success was greater for brown bear (0.88). Results for three species show that reliable individual genotyping, including sex identification, is now possible from eDNA in snow tracks, underlining its vast potential to complement the non-invasive genetic methods used for wildlife. To fully leverage the application of snow track eDNA, improved understanding of the ideal species- and site-specific sampling conditions, as well as laboratory methods promoting genotyping success are needed. This will also inform efforts to retrieve and type nuclear DNA from other eDNA samples, thereby advancing eDNA–based individual and population-level studies.

Methods
Snow tracks of brown bear, lynx and wolf were collected opportunistically during winter in 2019, 2020 and 2022 in the Slovenian Alps and Dinaric Mountains (seven brown bear samples and nine lynx samples) and in the French Alps (ten wolf samples). For genotyping of each species, we used a set of STR markers designed for optimal multiplex amplification and HTS genotyping. The brown bear set includes 13 STR recently described and used for individual profiling from fecal DNA, with the addition of a sex-specific marker. For wolf and lynx, we used 13 new STRs. We performed sequencing on a NovaSeq platform (2x150 bp) (libraries with id DIVJA088, DIVJA089, DAB074) and on a Miniseq platform (2x150 bp) (library LF22). Amplicons were sequenced in multiplexes, and necessary information for demultiplexing is in .ngsfilter files.

环境DNA（environmental DNA, eDNA）研究的持续进步，使得从eDNA样本中获取种内变异成为可能，为拓展野生动物种群的非侵入式遗传学研究开辟了全新机遇。然而，如同非侵入式遗传学中常规开展的那样，利用eDNA样本进行个体基因分型仍面临诸多挑战。本研究首次成功实现了从三种大型食肉动物的雪迹中获取的eDNA的个体基因分型，这三种食肉动物分别为棕熊（Ursus arctos）、欧亚猞猁（Lynx lynx）以及灰狼（Canis lupus）。研究采用针对水体eDNA分离的方案提取DNA，并通过高通量测序平台对短串联重复序列（short tandem repeats, STR）扩增子进行测序分型；针对棕熊，额外加入了性别标记进行分型。所有物种均成功获得了个体基因型，但不同样本及物种间的基因分型效果存在差异。棕熊样本的个体基因分型成功率（5/7）高于灰狼（7/10）与猞猁（4/9），但棕熊的位点分型成功率为0.88。三种物种的研究结果表明，如今可通过雪迹eDNA实现可靠的个体基因分型，包括性别鉴定，这凸显了其在补充野生动物非侵入式遗传研究方面的巨大潜力。若要充分发挥雪迹eDNA的应用价值，还需进一步明晰适配不同物种与采样位点的理想采样条件，以及提升基因分型成功率的实验室方法。这也将为从其他eDNA样本中获取并分型核DNA提供参考，从而推动基于eDNA的个体及种群水平研究发展。
方法
2019年、2020年及2022年冬季，我们在斯洛文尼亚阿尔卑斯山区与迪纳拉山脉（采集7份棕熊样本、9份猞猁样本）以及法国阿尔卑斯山区（采集10份灰狼样本），机会性收集了棕熊、猞猁与灰狼的雪迹样本。针对各物种的基因分型，我们采用了一组经过优化以实现高效多重扩增与高通量测序（high-throughput sequencing, HTS）分型的STR标记。棕熊的标记组包含13个此前已被报道并用于粪便DNA个体识别的STR位点，另加入了一个性别特异性标记。针对灰狼与猞猁，我们使用了13个全新的STR位点。测序工作分别在NovaSeq平台（2×150 bp，文库编号为DIVJA088、DIVJA089、DAB074）与Miniseq平台（2×150 bp，文库编号为LF22）上完成。扩增子以多重方式进行测序，解复用所需的相关信息存储于.ngsfilter格式文件中。