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Sci Rep. 2017 Jun 28;7(1):4339. doi: 10.1038/s41598-017-04511-0.

Method for preparing DNA from feces in guanidine thiocyanate solution affects 16S rRNA-based profiling of human microbiota diversity.

Author information

1
Laboratory of Vaccine Materials and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, 567-0085, Japan.
2
Department of Physical Activity Research, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Tokyo, 162-8636, Japan.
3
Laboratory of Bioinformatics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, 567-0085, Japan.
4
Department of Microbiology and Immunology, Kobe University Graduate School of Medicine, Hyogo, 650-0017, Japan.
5
Department of Physical Activity Research, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Tokyo, 162-8636, Japan. miyachi@nibiohn.go.jp.
6
Laboratory of Vaccine Materials and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, 567-0085, Japan. kunisawa@nibiohn.go.jp.
7
Department of Microbiology and Immunology, Kobe University Graduate School of Medicine, Hyogo, 650-0017, Japan. kunisawa@nibiohn.go.jp.
8
Graduate School of Medicine, Graduate School of Pharmaceutical Sciences, Graduate School of Dentistry, Osaka University, Osaka, 565-0871, Japan. kunisawa@nibiohn.go.jp.
9
Division of Mucosal Immunology, Department of Microbiology and Immunology and International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan. kunisawa@nibiohn.go.jp.

Abstract

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.

PMID:
28659635
PMCID:
PMC5489508
DOI:
10.1038/s41598-017-04511-0
[Indexed for MEDLINE]
Free PMC Article

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