DNA was extracted from ~80 mg of feces using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA, USA, D4300). The V3–V4 region of the bacterial 16S rRNA genes was amplified by PCR. Dual indices (barcodes) and Illumina sequencing adapters were added to the amplicons using the Nextera XT Index kit (Illumina, Inc., San Diego, CA, USA), followed by DNA purification (Agencourt AMPure XP, Beckman Coulter, Brea, CA, USA). Individual barcoded DNA samples were then quantified with Qubit dsDNA HS Assay kit with Qubit 2.0 (Thermo Fisher Scientific, Waltham, MA, USA), quantified with DNA 7500 kit