2.10. Fecal Pellet DNA Extraction and 16S rRNA Gene Sequencing and Analysis

Bacterial DNA was extracted from the feces using an E.Z.N.A^® Stool DNA kit (Omega Bio-Tek, Inc.; Norcross, GA, USA) and purified using magLEAD 12gc (Precision System Science Co., Ltd.; Chiba, Japan). PCR was performed using KAPA HiFi HotStart ReadyMix (Nippon Genetics Co.,Ltd.; Tokyo, Japan) and the primer set (forward: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′, and reverse: 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′) for the V3–V4 regions of 16S rRNA. The amplicons were purified using AMPure XP (Beckman Coulter; Brea, CA, USA). DNA from each sample was added to different index sequences using the Nextera XT index kit (Illumina; San Diego, CA, USA). Mixed samples were prepared by pooling approximately equal amounts of each amplified DNA and sequenced using Miseq Reagent Kit V3 (600 cycle) and a MiSeq sequencer (Illumina), in accordance with the manufacturer’s instructions.

The sequencing data were analyzed using Qiime2 (version 2020.11) [32]. To trim the primer region from the raw sequences, Cutadapt in the Qiime2 plugin was used (https://doi.org/10.14806/ej.17.1.200, accessed on 27 May 2021). The sequences without primer regions were processed for quality control, paired-end read joining, chimera filtering, and amplicon sequence varient (ASV) table construction using the DADA2 algorithm [33]. For each ASV-representative sequence, BLAST [34] was used to assign the taxonomy based on the SILVA database (version 138) [35]. After randomly sampling 4100 reads using a feature table [36], the compositional data were converted, and diversity analysis was performed.