Queensland Alliance for Agriculture and Food Innovation, Centre for Animal Science, The University of Queenslandgrid.1003.2, Brisbane, Queensland, Australia.
School of Veterinary Science, Faculty of Science, The University of Queenslandgrid.1003.2, Brisbane, Queensland, Australia.
Microbiol Spectr. 2022 Apr 27;10(2):e0041221. doi: 10.1128/spectrum.00412-21. Epub 2022 Apr 11.
The reproductive tract metagenome plays a significant role in the various reproductive system functions, including reproductive cycles, health, and fertility. One of the major challenges in bovine vaginal metagenome studies is host DNA contamination, which limits the sequencing capacity for metagenomic content and reduces the accuracy of untargeted shotgun metagenomic profiling. This is the first study comparing the effectiveness of different host depletion and DNA extraction methods for bovine vaginal metagenomic samples. The host depletion methods evaluated were slow centrifugation (Soft-spin), NEBNext Microbiome DNA Enrichment kit (NEBNext), and propidium monoazide (PMA) treatment, while the extraction methods were DNeasy Blood and Tissue extraction (DNeasy) and QIAamp DNA Microbiome extraction (QIAamp). Soft-spin and QIAamp were the most effective host depletion method and extraction methods, respectively, in reducing the number of cattle genomic content in bovine vaginal samples. The reduced host-to-microbe ratio in the extracted DNA increased the sequencing depth for microbial reads in untargeted shotgun sequencing. Bovine vaginal samples extracted with QIAamp presented taxonomical profiles which closely resembled the mock microbial composition, especially for the recovery of Gram-positive bacteria. Additionally, samples extracted with QIAamp presented extensive functional profiles with deep coverage. Overall, a combination of Soft-spin and QIAamp provided the most robust representation of the vaginal microbial community in cattle while minimizing host DNA contamination. In addition to the host tissue collected during the sampling process, bovine vaginal samples are saturated with large amounts of extracellular DNA and secreted proteins that are essential for physiological purposes, including the reproductive cycle and immune defense. Due to the high host-to-microbe genome ratio, which hampers the sequencing efficacy for metagenome samples and the recovery of the actual metagenomic profiles, bovine vaginal samples cannot benefit from the full potential of shotgun sequencing. This is the first investigation on the most effective host depletion and extraction methods for bovine vaginal metagenomic samples. This study demonstrated an effective combination of host depletion and extraction methods, which harvested higher percentages of 16S rRNA genes and microbial reads, which subsequently led to a taxonomical profile that resembled the actual community and a functional profile with deeper coverage. A representative metagenomic profile is essential for investigating the role of the bovine vaginal metagenome for both reproductive function and susceptibility to infections.
生殖道宏基因组在各种生殖系统功能中发挥着重要作用，包括生殖周期、健康和生育能力。牛阴道宏基因组研究的主要挑战之一是宿主 DNA 污染，这限制了宏基因组内容的测序能力，并降低了非靶向 shotgun 宏基因组分析的准确性。这是第一项比较不同宿主去除和 DNA 提取方法对牛阴道宏基因组样品效果的研究。评估的宿主去除方法包括低速离心（Soft-spin）、NEBnext 微生物 DNA 富集试剂盒（NEBnext）和吖啶单甲醚（PMA）处理，而提取方法为 DNeasy Blood 和 Tissue 提取试剂盒（DNeasy）和 QIAamp DNA 微生物提取试剂盒（QIAamp）。Soft-spin 和 QIAamp 分别是减少牛阴道样本中牛基因组含量最有效的宿主去除方法和提取方法。提取 DNA 中减少的宿主与微生物比例增加了非靶向 shotgun 测序中微生物读数的测序深度。用 QIAamp 提取的牛阴道样本呈现的分类群谱与模拟微生物组成非常相似，尤其是恢复革兰氏阳性菌。此外，用 QIAamp 提取的样本具有广泛的功能谱，深度覆盖。总体而言，Soft-spin 和 QIAamp 的组合提供了牛阴道微生物群落最稳健的代表性，同时最大限度地减少了宿主 DNA 污染。除了在采样过程中收集的宿主组织外，牛阴道样本还充满了大量的细胞外 DNA 和分泌蛋白，这些蛋白质对于生理目的，包括生殖周期和免疫防御，是必不可少的。由于宿主与微生物基因组的比例很高，这阻碍了宏基因组样本的测序效果和实际宏基因组谱的恢复，牛阴道样本无法充分发挥 shotgun 测序的潜力。这是第一项关于牛阴道宏基因组样品最有效宿主去除和提取方法的研究。本研究证明了宿主去除和提取方法的有效结合，可以收获更高比例的 16S rRNA 基因和微生物读数，从而导致与实际群落相似的分类群谱和具有更深覆盖度的功能谱。代表性宏基因组谱对于研究牛阴道宏基因组在生殖功能和感染易感性方面的作用至关重要。
