Schellenberg John J, Oh Angela Yena, Hill Janet E
Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada.
Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada.
J Microbiol Methods. 2017 May;136:57-64. doi: 10.1016/j.mimet.2017.03.003. Epub 2017 Mar 9.
The vaginal microbiome is increasingly characterized by deep sequencing of universal genes. However, there are relatively few studies of how different specimen collection and sample storage and processing influence these molecular profiles. Here, we evaluate molecular microbial community profiles of samples collected using the HerSwab™ self-sampling device, compared to nylon swabs and under different storage conditions. In order to minimize technical variation, mixtures of 11 common vaginal bacteria in simulated vaginal fluid medium were sampled and DNA extracts prepared for massively parallel sequencing of the cpn60 universal target (UT). Three artificial mixtures imitating commonly observed vaginal microbiome profiles were easily distinguished and proportion of sequence reads correlated with the estimated proportion of the organism added to the artificial mixtures. Our results indicate that cpn60 UT amplicon sequencing quantifies the proportional abundance of member organisms in these artificial communities regardless of swab type or storage conditions, although some significant differences were observed between samples that were stored frozen and thawed prior to DNA extraction, compared to extractions from samples stored at room temperature for up to 7days. Our results indicate that an on-the-market device developed for infectious disease diagnostics may be appropriate for vaginal microbiome profiling, an approach that is increasingly facilitated by rapidly dropping deep sequencing costs.
阴道微生物群越来越多地通过对通用基因的深度测序来表征。然而,关于不同的样本采集、样本储存和处理方式如何影响这些分子图谱的研究相对较少。在此,我们评估了使用HerSwab™ 自采样装置采集的样本的分子微生物群落图谱,并与尼龙拭子在不同储存条件下的情况进行了比较。为了尽量减少技术差异,我们对模拟阴道液培养基中的11种常见阴道细菌混合物进行了采样,并制备了DNA提取物,用于对cpn60通用靶标(UT)进行大规模平行测序。三种模仿常见阴道微生物群图谱的人工混合物很容易区分,并且序列读数的比例与添加到人工混合物中的生物体的估计比例相关。我们的结果表明,cpn60 UT扩增子测序能够量化这些人工群落中成员生物体的相对丰度,而不受拭子类型或储存条件的影响,尽管在DNA提取前经过冷冻和解冻的样本与在室温下储存长达7天的样本提取物之间观察到了一些显著差异。我们的结果表明,一种为传染病诊断开发的市售装置可能适用于阴道微生物群分析,随着深度测序成本的迅速下降,这种方法越来越容易实现。