DNA extraction, library preparation, and 16S rRNA gene amplicon sequencing

Stool specimens were thawed at 4 °C and were diluted 1:3 with milliQ water to create a stool slurry. DNA was isolated using the ZR-96 Fecal DNA Kit (Zymo Research, Irvine, CA) following a validated protocol [20]. The Illumina MiSeq sequencing library preparation protocol for 16S rRNA gene amplicons was followed with modifications [20]. Briefly, the 16S rRNA V4 region was amplified using primers 515fXT (GTGBCAGCMGCCGCGGTAA) and 806rXT (GGACTACHVGGGTWTCTAAT). Quality control, quantification, normalization, pooling, and sequencing of the library was performed as previously described [20]. Approximately 11 pM of the pooled, multiplexed samples were mixed with 37.5% PhiX spike-in control DNA and sequenced on an Illumina MiSeq instrument to generate 2 × 300 bp reads. Four MiSeq runs were performed with 56 multiplexed samples per run. In total, 224 samples were sequenced including technical replicates, mock communities of known composition (HM-782D; BEI Resources, Manassas, VA) and no-template controls.