Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis

Background
Molecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species.
Results
The total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001).
Conclusions
The results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal microbiota studies employing different sampling strategies should be comparable in the absence of other technical confounders. The Evalyn® self-sampling device performed equally well compared to samples taken by a clinician, and hence offers a good-quality microbiota sample without the need for a gynecological examination. The amount of collected sample as well as the DNA and protein yield varied across the sampling techniques, which may have practical implications for study design.

研究背景
下一代测序（next-generation sequencing）等分子生物学方法正被广泛用于表征健康与疾病状态下的阴道微生物群（vaginal microbiota）。既往研究多聚焦于微生物群的生物学变异特征，而对于采样相关因素如何影响实验结果的认知仍较为匮乏。本研究旨在探讨采样装置与解剖采样部位对阴道微生物群研究相关定量与定性结果的影响。我们纳入10名具有多样化临床特征的芬兰女性，分别使用植绒拭子（flocked swabs）、Evalyn®自采样装置、无菌塑料刮匙以及宫颈刷，从阴道穹窿、阴道壁及宫颈采集样本。基于16S rRNA基因（16S rRNA gene）的Illumina MiSeq测序（Illumina MiSeq sequencing）结果，我们对样本的DNA与蛋白得率、细菌载量、微生物群多样性及物种组成进行了比较分析。我们量化了采样变量与内在变量在微生物群整体变异中的相对贡献，并采用多种常用指标对微生物群特征进行评估，包括α多样性（alpha diversity）、β多样性（beta diversity）以及主要细菌属与物种的丰度。
研究结果
总DNA得率强烈依赖于采样装置，且受采样解剖部位的影响相对较小。采样策略对蛋白得率与细菌载量无显著影响。基于MiSeq测序结果，所有受试采样方法获得的微生物群特征具有高度一致性。采样方法仅能解释2%的微生物群整体变异（p值=0.89），远低于临床状态等内在因素的贡献：细菌性阴道病（bacterial vaginosis）显微镜检测相关因素占53%（p=0.0001）、出血相关因素占19%（p=0.0001），以及个体间变异占11%（p值=0.0001）。
研究结论
本研究结果表明，不同采样策略可获得具有可比性的阴道微生物群组成与多样性特征。因此，在不存在其他技术混杂因素的前提下，既往及未来采用不同采样策略开展的阴道微生物群研究结果应具有可比性。Evalyn®自采样装置的检测效果与临床医师采集的样本相当，因此无需进行妇科检查即可获得高质量的微生物群样本。不同采样技术的样本采集量、DNA与蛋白得率存在差异，这对研究设计具有实际指导意义。