Abstract：
ObjectivesWe evaluated how storing vaginal samples at room temperature in stabilising solutions versus immediate freezing affects 16S rRNA gene amplicon sequencing-based microbiota studies, aiming to simplify home and field collection.
METHODS: Twenty participants self-collected six mid-vaginal swabs that were stored in two nucleic acid preservatives (three in modified Solution C2 (Qiagen) and three in Amies/RNALater (Sigma)) in January-February 2016. From each set, two were immediately frozen (-80°C) and one was shipped to the University of Idaho (Moscow, Idaho) with return shipping to the Institute for Genome Sciences (Baltimore, Maryland). Amplicon sequencing of the 16S rRNA gene was used to characterise the vaginal microbiota, VALENCIA was used to assign community state types (CSTs), and quantitative PCR (qPCR) of 16S rRNA genes was used to estimate bacterial abundance. Cohen\'s Kappa statistic was used to assess within-participant agreement. Bayesian difference of means models assessed within-participant comparisons between shipped and immediately frozen samples.
RESULTS: There were 115 samples available for analysis. Average duration of transit for shipped samples was 8 days (SD: 1.60, range: 6-11). Within-participant comparisons of CSTs between shipped and immediately frozen samples revealed complete concordance (kappa: 1.0) for both preservative solutions. No significant differences comparing shipped and immediately frozen samples were found with taxon-level comparisons or bacterial abundances based on pan-bacterial qPCR.
CONCLUSIONS: Short-term room temperature shipping of vaginal swabs placed in stabilising solutions did not affect vaginal microbiota composition. Home collection with mail-in of vaginal samples may be a reasonable approach for research and clinical purposes to assess the vaginal microbiota.
METHODS: Twenty participants self-collected six mid-vaginal swabs that were stored in two nucleic acid preservatives (three in modified Solution C2 (Qiagen) and three in Amies/RNALater (Sigma)) in January-February 2016. From each set, two were immediately frozen (-80°C) and one was shipped to the University of Idaho (Moscow, Idaho) with return shipping to the Institute for Genome Sciences (Baltimore, Maryland). Amplicon sequencing of the 16S rRNA gene was used to characterise the vaginal microbiota, VALENCIA was used to assign community state types (CSTs), and quantitative PCR (qPCR) of 16S rRNA genes was used to estimate bacterial abundance. Cohen\'s Kappa statistic was used to assess within-participant agreement. Bayesian difference of means models assessed within-participant comparisons between shipped and immediately frozen samples.
RESULTS: There were 115 samples available for analysis. Average duration of transit for shipped samples was 8 days (SD: 1.60, range: 6-11). Within-participant comparisons of CSTs between shipped and immediately frozen samples revealed complete concordance (kappa: 1.0) for both preservative solutions. No significant differences comparing shipped and immediately frozen samples were found with taxon-level comparisons or bacterial abundances based on pan-bacterial qPCR.
CONCLUSIONS: Short-term room temperature shipping of vaginal swabs placed in stabilising solutions did not affect vaginal microbiota composition. Home collection with mail-in of vaginal samples may be a reasonable approach for research and clinical purposes to assess the vaginal microbiota.
摘要：
我们评估了在室温下稳定溶液中储存阴道样品与立即冷冻对16SrRNA基因扩增子测序微生物群研究的影响。旨在简化家庭和现场收集。
方法：2016年1月至2月，20名参与者自行收集了6个阴道中部拭子，这些拭子储存在两种核酸防腐剂中（三种在改良溶液C2（Qiagen）中，三种在Amies/RNALater（Sigma）中）。从每一组，两个被立即冷冻（-80°C），一个被运送到爱达荷州大学（莫斯科，爱达荷州）与返回寄往基因组科学研究所（巴尔的摩，马里兰)。16SrRNA基因的扩增子测序用于表征阴道微生物群，瓦伦西亚被用来分配社区状态类型(CST)，和16SrRNA基因的定量PCR（qPCR）用于估计细菌丰度。科恩的Kappa统计数据用于评估参与者内部协议。均值模型的贝叶斯差异评估了已装运样品和立即冷冻样品之间的参与者内部比较。
结果：有115个样品可供分析。运输样品的平均运输持续时间为8天(SD:1.60,范围:6-11)。参与者内部比较运输样品和立即冷冻样品之间的CST显示两种防腐剂溶液完全一致（κ：1.0）。使用基于泛细菌qPCR的分类单元水平比较或细菌丰度，未发现运输和立即冷冻的样品的显着差异。
结论：放置在稳定溶液中的阴道拭子的短期室温运输不会影响阴道微生物群组成。通过邮寄阴道样本进行家庭收集可能是评估阴道微生物群的研究和临床目的的合理方法。
方法：2016年1月至2月，20名参与者自行收集了6个阴道中部拭子，这些拭子储存在两种核酸防腐剂中（三种在改良溶液C2（Qiagen）中，三种在Amies/RNALater（Sigma）中）。从每一组，两个被立即冷冻（-80°C），一个被运送到爱达荷州大学（莫斯科，爱达荷州）与返回寄往基因组科学研究所（巴尔的摩，马里兰)。16SrRNA基因的扩增子测序用于表征阴道微生物群，瓦伦西亚被用来分配社区状态类型(CST)，和16SrRNA基因的定量PCR（qPCR）用于估计细菌丰度。科恩的Kappa统计数据用于评估参与者内部协议。均值模型的贝叶斯差异评估了已装运样品和立即冷冻样品之间的参与者内部比较。
结果：有115个样品可供分析。运输样品的平均运输持续时间为8天(SD:1.60,范围:6-11)。参与者内部比较运输样品和立即冷冻样品之间的CST显示两种防腐剂溶液完全一致（κ：1.0）。使用基于泛细菌qPCR的分类单元水平比较或细菌丰度，未发现运输和立即冷冻的样品的显着差异。
结论：放置在稳定溶液中的阴道拭子的短期室温运输不会影响阴道微生物群组成。通过邮寄阴道样本进行家庭收集可能是评估阴道微生物群的研究和临床目的的合理方法。
