Graeber Enid, Tysha Alona, Nisar Azlan, Wind Daniel, Mendling Werner, Finzer Patrick, Dilthey Alexander
Institute of Medical Microbiology and Hospital Hygiene, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
German Center for Infections in Obstetrics and Gynecology, Wuppertal, Germany.
BMC Microbiol. 2025 Aug 25;25(1):544. doi: 10.1186/s12866-025-04236-5.
The vaginal microbiome plays an important role in female health; it is associated with reproductive success, susceptibility to sexually transmitted infections, and, importantly, the most prevalent vaginal condition in reproduction-age women, bacterial vaginosis (BV). Traditionally, 16S rRNA gene sequencing-based approaches have been used to characterize the composition of vaginal microbiomes, but shallow shotgun metagenomic sequencing (SMS) approaches, in particular when implemented with the Oxford Nanopore Technologies, have important potential advantages with respect to cost effectiveness, speed of data generation, and the availability of flexible multiplexing schemes.
Based on a study cohort of n = 52 women, of which 23 were diagnosed with BV, we evaluated the applicability of Nanopore-based SMS for the characterization of vaginal microbiomes in direct comparison to Illumina 16S-based sequencing. We observed perfect agreement between the two approaches with respect to detecting the dominance of individual samples by either Lactobacilli, vaginosis-associated, or other taxa; very high concordance (92%) with respect to community state type (CST) classification; and a high degree of concordance with respect to the overall clustering structures of the sequenced microbiomes. Comparing the inferred abundances of individual species in individual samples, we observed significant differences (Wilcoxon signed-rank test p < 0.05) between the two approaches for 12 of the 20 species most abundant in our cohort, indicating differences in the fine-scale characterization of vaginal microbiomes. Higher overall abundance of Gardnerella vaginalis, associated with an increased number of CST IV detections, in the Nanopore shallow SMS data indicated potentially increased sensitivity of this approach to dysbiotic states of the vaginal microbiome. Nanopore shallow SMS also enabled the methylation-based quantification of different human cell types in the characterized samples as well as the detection of non-prokaryotic species, including Lactobacillus phage and Candida albicans in study participants with microscopically detected Candida. One important potential limitation of the evaluated Nanopore-based SMS approach was marked variation in sequencing yields.
Our study demonstrated the successful application and potential advantages of Nanopore-based shallow SMS for the characterization of vaginal microbiomes and paves the way for its application in larger-scale research or diagnostic settings.
阴道微生物群在女性健康中起着重要作用；它与生殖成功率、性传播感染易感性相关，重要的是，还与育龄女性中最常见的阴道疾病——细菌性阴道病（BV）有关。传统上，基于16S rRNA基因测序的方法已被用于表征阴道微生物群的组成，但浅层鸟枪法宏基因组测序（SMS）方法，特别是与牛津纳米孔技术一起实施时，在成本效益、数据生成速度和灵活的多重测序方案可用性方面具有重要的潜在优势。
基于一个n = 52名女性的研究队列，其中23名被诊断为BV，我们评估了基于纳米孔的SMS在表征阴道微生物群方面的适用性，并与基于Illumina 16S的测序进行了直接比较。我们观察到，在检测单个样本中乳酸杆菌、阴道病相关或其他分类群的优势方面，两种方法完全一致；在社区状态类型（CST）分类方面，一致性非常高（92%）；在测序微生物群的总体聚类结构方面，一致性程度很高。比较单个样本中单个物种的推断丰度，我们发现，在我们队列中最丰富的20个物种中的12个物种上，两种方法之间存在显著差异（Wilcoxon符号秩检验p < 0.05），这表明在阴道微生物群的精细表征方面存在差异。纳米孔浅层SMS数据中阴道加德纳菌的总体丰度较高，与CST IV检测数量增加相关，这表明该方法对阴道微生物群生态失调状态的敏感性可能增加。纳米孔浅层SMS还能够对特征样本中不同人类细胞类型进行基于甲基化的定量，以及检测非原核生物物种，包括在显微镜下检测到念珠菌的研究参与者中的乳酸杆菌噬菌体和白色念珠菌。所评估的基于纳米孔的SMS方法的一个重要潜在局限性是测序产量存在显著差异。
我们的研究证明了基于纳米孔的浅层SMS在表征阴道微生物群方面的成功应用和潜在优势，并为其在大规模研究或诊断环境中的应用铺平了道路。
