2.2.8. Analysis of Fecal Microbiota
Fecal samples were collected by participants themselves using a collection kit (Kyoto Institute of Nutrition & Pathology, Inc., Kyoto, Japan) and suspended in DESS buffer (0.25 M disodium ethylenediaminetetraacetic acid pH 8.0, 20% dimethyl sulfoxide, NaCl saturated) [The V3 and V4 regions of the 16S rRNA gene amplicons were prepared according to the 16S Metagenomic Sequencing Library Preparation (Illumina, San Diego, CA, USA), and sequencing was conducted using the Illumina Miseq system (Illumina). After sequencing, the obtained fastq files were analyzed using Quantitative Insights into Microbial Ecology 2 (QIIME2). The SILVA database (ver. 138) was used for taxonomic classification. The relative abundance (genus level) was calculated, and analyzed for genera that showed more than 0.01% mean relative abundance. The genus Moreover, k-means clustering based on the composition of the fecal microbiota (0 week) was performed, and the fecal microbiome of participants was divided into three clusters (k = 3). We used EZR software version 1.40 to perform k-means clustering. The initial seed value was set to 10. The genera detected frequently (more than 50% of the samples) were selected and used for the clustering. In addition, the genera not selected were classified together as “other genera”.