Fecal 16S rRNA Sequencing with Bead-Beating

The fecal sample mixture was mechanically disrupted using the bead-beating method. DNA was extracted using Gene Prep Star PI-80X (Kurabo Industries, Osaka, Japan). After DNA extraction, the V3-V4 region of the 16S rRNA gene was amplified using the following primers: forward, 5′-TCG GCA GCG TCA GAT GTG TAT AAG CGA CAG CCT ACG GGN GGC WGC AG-3′; reverse, 5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′ [30 (link)]. Amplicons were sequenced via the paired-end method using MiSeq (Illumina, San Diego, CA, USA). The overall procedure from fecal sampling to 16S rRNA sequencing was performed according to a previously described protocol [31 (link)].