Method for preparing DNA from feces in guanidine thiocyanate solution affects 16S rRNA-based profiling of human microbiota diversity.
Method for preparing DNA from feces in guanidine thiocyanate solution affects 16S rRNA-based profiling of human microbiota diversity.
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DOI：
10.1038/s41598-017-04511-0
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发表时间：
2017-06-28
影响因子：
4.6
通讯作者：
Kunisawa J
中科院分区：
文献类型：
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作者：
Hosomi K;Ohno H;Murakami H;Natsume-Kitatani Y;Tanisawa K;Hirata S;Suzuki H;Nagatake T;Nishino T;Mizuguchi K;Miyachi M;Kunisawa J
基于16S rRNA基因的宏基因组分析通常用于检查粪便中共生细菌的多样性和丰度，目前已知粪便中共生细菌与人类健康和疾病相关。与冷冻方法相比，使用硫氰酸胍 (GuSCN) 溶液作为一种不太繁琐的方法在室温下运输和储存粪便样本以进行 DNA 分析；然而，测量 GuSCN 溶液中粪便细菌成分的最佳方法仍有待研究。在这里，我们研究了各种因素的影响，例如预处理（例如，机械裂解前去除GuSCN溶液并用磷酸盐缓冲盐水（PBS）洗涤粪便）、GuSCN溶液中的粪便浓度、储存时间和粪便二次取样的位置对GuSCN溶液中基于16S rRNA的粪便细菌分析的影响。我们发现预处理和粪便浓度影响细菌组成，并且二次采样位置略有变化。基于这些结果，我们提出了一个基本方案，包括粪便取样、样品储存和 DNA 提取，用于基于 16S rRNA 的悬浮在 GuSCN 溶液中的粪便中的细菌成分分析。
Metagenomic analysis based on the 16S rRNA gene is generally performed to examine the diversity and abundance of commensal bacteria in feces, which is now recognized to be associated with human health and diseases. Guanidine thiocyanate (GuSCN) solution is used as a less onerous way compared with a frozen method to transport and stock fecal samples at room temperature for DNA analysis; however, optimal methods to measure fecal bacterial composition in GuSCN solution remain to be investigated. Here, we examined the influence of various factors such as pretreatment (e.g., removing GuSCN solution and washing feces with phosphate-buffered saline (PBS) before mechanical lysis), fecal concentration in the GuSCN solution, storage time, and position of fecal subsampling on the 16S rRNA-based analysis of fecal bacteria in GuSCN solution. We found that pretreatment and fecal concentration affected the bacterial composition, and a little change was noted with subsampling position. Based on these results, we propose a basic protocol, including fecal sampling, sample storage, and DNA extraction, for the 16S rRNA-based analysis of bacterial composition in feces suspended in GuSCN solution.