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.
Highlights
Gut commensal bacteria are recognized as an important factor in the maintenance of health because they facilitate food digestion and concomitant metabolite generation[1], regulate the immune system[2], and inhibit infection by potentially pathogenic microorganisms by competing for niches[3]
GuSCN solution is used as a simple way to transport and stock fecal samples at room temperature for DNA analysis; optimal methods for extraction of DNA from fecal samples in GuSCN solution to measure fecal bacterial composition remain to be investigated
We initially investigated whether pretreatment or no treatment before mechanical lysis affected microbial composition in feces suspended in GuSCN solution
Summary
Gut commensal bacteria are recognized as an important factor in the maintenance of health because they facilitate food digestion and concomitant metabolite generation[1], regulate the immune system[2], and inhibit infection by potentially pathogenic microorganisms by competing for niches[3]. Including sample storage (frozen or room temperature; solution composition), homogenization method for DNA extraction (e.g., mechanical or enzymatic)[11, 12], and choice of PCR primers[13].
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