title: 16S rRNA gene amplicon sequencing (V3-V4 region)
design: DNA was extracted from 80 mg of feces (QIAamp PowerFecal DNA Kit); PCR amplification; single amplicons (460 bp) were cleaned up using AMPure XP beads (Agencourt AMPure XP); Dual indices (barcodes) and Illumina sequencing adapters were added using the Nextera XT Index kit; DNA purification (Agencourt AMPure XP); Individual barcoded DNA samples were quantified (Qubit dsDNA HS Assay Kit with Qubit 2.0; Thermo Fisher Scientific), qualified (DNA 1000 Kit with Agilent 2100 Bioanalyzer), normalized, and pooled. Multiplexed libraries, were diluted to 13 pM and denatured with NaOH prior to sequencing on the MiSeq system (Illumina) using the MiSeq reagent Kit v3 600 cycle (2  300 bp; Illumina).