*2.3. Microbial-Community Analysis*

A polymerase chain reaction (PCR) was carried out using primers specific to the V3–V4 region involving pyrosequencing tags of the 16S ribosomal RNA (rRNA) gene. The taxonomic classification of each read was assigned on the basis of a search of the EzBioCloud 16S database [38,39]. By applying the data from this database, a hierarchical taxonomic classification was obtained [32,37]. These analyses were carried out by Chun Lab (Seoul, Korea).

#### *2.4. Bioinformatics Analysis*

The relative abundance of the 16S rRNA gene for each operational taxonomic unit (OTU) was used to determine the absolute abundance of each OTU by multiplying the respective relative abundance by the total number of 16S rRNA gene copies. The Microbiome and Phyloseq packages in R software version 3.50 (Lucent Technologies, Murray Hill, NJ, USA) were used for analysis [40]. Heatmaps and core heatmaps [41] were used for visualization. Core line-plot and t-distributed stochastic neighbor embedding (t-SNE) [42] analyses were performed using the Microbiome Rtsne and Vegan packages.
