TY - JOUR
T1 - Method for preparing DNA from feces in guanidine thiocyanate solution affects 16S rRNA-based profiling of human microbiota diversity
AU - Hosomi, Koji
AU - Ohno, Harumi
AU - Murakami, Haruka
AU - Natsume-Kitatani, Yayoi
AU - Tanisawa, Kumpei
AU - Hirata, Soichiro
AU - Suzuki, Hidehiko
AU - Nagatake, Takahiro
AU - Nishino, Tomomi
AU - Mizuguchi, Kenji
AU - Miyachi, Motohiko
AU - Kunisawa, Jun
N1 - Funding Information:
This work was supported by grants from the Ministry of Health, Labour and Welfare of Japan; and the Japan Agency for Medical Research and Development (16817372, 16768433, 16727634 and 17933231); the Ministry of Education, Culture, Sports, Science and Technology of Japan [grant numbers 26293111, 16H01373, 23229004, 15K18950, 17918005 and 15638619]; the Astellas Foundation for Research on Metabolic Disorders; the Terumo Foundation for Life Sciences and Arts; the Suzuken Memorial Foundation; Nipponham Foundation for the Future of Food; and the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry.
Publisher Copyright:
© The Author(s) 2017.
PY - 2017/12/1
Y1 - 2017/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41598-017-04511-0
DO - 10.1038/s41598-017-04511-0
M3 - Article
C2 - 28659635
AN - SCOPUS:85021672997
SN - 2045-2322
VL - 7
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 4339
ER -