Llarena Ann-Katrin, Haverkamp Thomas H A, Gulliksen Wenche Støldal, Herstad Kristin, Holst-Jensen Arne, Skjerve Eystein, Rannem Lisbeth, Rodriguez-Campos Sabrina, Øines Øivind
Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Ås, Norway.
Department of Animal Health, Welfare and Food Safety, Norwegian Veterinary Institute, Ås, Norway.
PLoS One. 2025 May 12;20(5):e0313808. doi: 10.1371/journal.pone.0313808. eCollection 2025.
Collecting fecal samples using dry preservatives is an attractive option in large epidemiological studies as they are easy to use, cheap and independent of cold chain logistics. Here, we test four DNA extraction methods with the aim of identifying an efficient procedure to extract high-quality DNA from fecal material of canine, sheep, equine, bovine, and pig collected on dry blood spot cards, with the goal of generating good quality shotgun metagenomics datasets. Further, the suitability of Illumina shotgun metagenomic sequencing at 20 million paired-end (PE) read depth per sample was assessed on its ability to successfully characterize the taxonomic and functional aspects of the resulting fecal microbiome.
DNA was extracted from pig feces and mock communities collected on blood spot cards using four DNA extraction methods; two different methods of the QIAsymphony® PowerFecal® Pro DNA Kit, the ZymoBIOMICS™ DNA Miniprep Kit, and the MagNA Pure 96 DNA and Viral NA Small Volume Kit. Possible extraction bias was controlled by amplicon sequencing of mock communities. Fecal samples from canine, sheep, equine, bovine, and pig were thereafter subjected to the best performing DNA extraction method and shotgun metagenomic sequencing to determine sequencing efforts for functional and taxonomic analysis.
The four DNA extraction methods demonstrated similar community composition in the sequenced bacterial mock community. The QIAsymphony® PowerFecal® Pro DNA Kit was identified as the DNA extraction method of choice, and the resulting DNA was subjected to shotgun metagenomic sequencing with 20million PE reads. We found that higher number of reads increased the richness of observed genera between 100,000 and 5 million reads, after which higher sequencing effort did not increase the richness of the metagenomes. As for functional analysis, the number of low abundance functions in the metagenomes of the animals' feces increased with sequencing depth above 20 million PE reads.
Our experiments identified several methods suitable for extracting DNA from feces collected on blood spot cards. The QIAGEN's Blood and Tissue kit on the QiaSymphony platform fulfilled the criteria of high yield, quality, and unbiased DNA, while maintaining high throughput for shotgun metagenomic sequencing. A sequencing depth of 20 million PE reads proved adequate for taxonomic estimations and identifying common functional pathways. Detecting rarer traits, however, requires more sequencing effort.
在大型流行病学研究中，使用干式防腐剂收集粪便样本是一个颇具吸引力的选择，因为它们易于使用、成本低廉且不受冷链物流的限制。在此，我们测试了四种DNA提取方法，旨在确定一种高效的程序，以便从收集在干血斑卡上的犬、羊、马、牛和猪的粪便样本中提取高质量的DNA，目标是生成高质量的鸟枪法宏基因组数据集。此外，还评估了在每个样本2000万对端（PE）读长深度下进行Illumina鸟枪法宏基因组测序，以成功表征所得粪便微生物组的分类学和功能方面的能力。
使用四种DNA提取方法从收集在血斑卡上的猪粪便和模拟群落中提取DNA；QIAsymphony® PowerFecal® Pro DNA试剂盒的两种不同方法、ZymoBIOMICS™ DNA微量制备试剂盒以及MagNA Pure 96 DNA和病毒核酸小体积试剂盒。通过对模拟群落进行扩增子测序来控制可能的提取偏差。此后，对犬、羊、马、牛和猪的粪便样本采用性能最佳的DNA提取方法并进行鸟枪法宏基因组测序，以确定用于功能和分类分析的测序工作量。
四种DNA提取方法在测序的细菌模拟群落中显示出相似的群落组成。QIAsymphony® PowerFecal® Pro DNA试剂盒被确定为首选的DNA提取方法，所得DNA进行了2000万PE读长的鸟枪法宏基因组测序。我们发现，在100,000至500万读长之间，更多的读长增加了观察到的属的丰富度，此后更高的测序工作量并未增加宏基因组的丰富度。至于功能分析，动物粪便宏基因组中低丰度功能的数量随着测序深度超过2000万PE读长而增加。
我们的实验确定了几种适合从收集在血斑卡上的粪便中提取DNA的方法。QiaSymphony平台上的QIAGEN血液和组织试剂盒满足了高产、高质量和无偏差DNA的标准，同时保持了鸟枪法宏基因组测序的高通量。2000万PE读长的测序深度被证明足以进行分类学估计和识别常见的功能途径。然而，检测更罕见的特征需要更多的测序工作量。
