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

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.

Association between functional lactase variants and a high abundance of Bifidobacterium in the gut of healthy Japanese people

“…A total of 9,285,977 high-quality paired sequences were obtained from 1,068 samples, and 8,695±2,255 (average±standard deviation) reads per sample were generated. The average of Bifidobacterium abundance was 6.3±7.8%, which was lower than that in previous Japanese reports (17.9±15.2%[ 3 ], 10.5±11.0%[ 4 ], 13.6±17.7%[ 5 ]). This discrepancy might be due to differences in faecal samplings, DNA extraction and analysis methods including the primers used[ 5 , 18 , 19 ], in addition to the distribution of the population in each study.…”

“…The underlying causes of long life in Japan have been debated, and the possibilities include good hygiene, a high level of health consciousness in Japan, and the Japanese diet[ 2 ]. In previous reports, Japanese healthy adults exhibited a higher abundance of Bifidobacterium compared to people from other countries[ 3 – 5 ]. Considering that Bifidobacterium naturally inhabits the human gastrointestinal tract (GIT) and is thought to play pivotal roles in maintaining human health[ 6 ], the higher abundance of this genus seems to be another potential reason for long life in Japan.…”

Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota

“…It should also be noted that other storage solutions, i.e . those using guanidine thiocyanate have been reported for microbiome surveys of the cervix (81) and feces (82). A weakness of the current study is that we did not examine the reproducibility of our results as each sample was extracted using each kit once.…”

Comprehensive Analysis of Gut Microbiota of a Healthy Population and Covariates Affecting Microbial Variation in Two Large Japanese Cohorts

“…The V3-V4 region of the 16S rRNA gene was ampli ed using KOD-Plus-v2 (Toyobo, Osaka, Japan) and sequenced by paired-end method using Illumina MiSeq instrument and the MiSeq v3 Reagent Kit (Illumina, San Diego, CA, United States). All the steps from fecal sampling to 16S rRNA gene amplicon sequencing was performed according to a previously described protocol [37].…”