The vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment but also to the use of conflicting methodologies to study its microbial composition. Here, we systematically assessed best practices for the sequencing-based characterization of the human vaginal microbiome. As far as 16S rRNA gene sequencing is concerned, the V1-V3 region performed best in silico, but limitations of current sequencing technologies meant that the V3-V4 region performed equally well. Both approaches presented very good agreement with qPCR quantification of key taxa, provided that an appropriate bioinformatic pipeline was used. Shotgun metagenomic sequencing presents an interesting alternative to 16S rRNA gene amplification and sequencing but requires deeper sequencing and more bioinformatic expertise and infrastructure. We assessed different tools for the removal of host reads and the taxonomic annotation of metagenomic reads, including a new, easy-to-build and -use reference database of vaginal taxa. This curated database performed as well as the best-performing previously published strategies. Despite the many advantages of shotgun sequencing, none of the shotgun approaches assessed here agreed with the qPCR data as well as the 16S rRNA gene sequencing.

中文翻译：

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用于基于序列表征人类阴道微生物组的体外和计算机实验方案的评估

阴道微生物组与广泛的健康结果有关。这导致了蓬勃发展的研究环境，但也导致了使用相互矛盾的方法来研究其微生物组成。在这里，我们系统地评估了基于测序的人类阴道微生物组表征的最佳实践。就 16S rRNA 基因测序而言，V1-V3 区域在 silico 中表现最佳，但当前测序技术的局限性意味着 V3-V4 区域表现同样出色。如果使用适当的生物信息学管道，两种方法都与关键分类群的 qPCR 定量非常一致。鸟枪法宏基因组测序是 16S rRNA 基因扩增和测序的有趣替代方案，但需要更深入的测序和更多的生物信息学专业知识和基础设施。我们评估了用于删除宿主读取和宏基因组读取的分类注释的不同工具，包括一个新的、易于构建和使用的阴道分类群参考数据库。这个精心策划的数据库的表现与之前发布的表现最佳的策略一样。尽管霰弹枪测序有许多优点，