title: 16S sequencing of fecal microbiota
design: The V4 hypervariable region of the 16S rRNA gene was amplified using uniquely barcoded 515F (forward) and 806R (reverse) sequencing primers to allow for multiplexing. Amplification reactions were performed using 12.5 uL of KAPA2G Robust HotStart ReadyMix (KAPA Biosystems), 1.5 uL of 10 uM forward and reverse primers, 7.5 uL of sterile water and 2 uL of DNA. The V4 region was amplified by cycling the reaction at 95C for 3 minutes, 18x cycles of 95C for 15 seconds, 50C for 15 seconds and 72C for 15 seconds, followed by a 5-minute 72C extension. All amplification reactions were done in duplicate to reduce amplification bias, pooled, and checked on a 1% agarose TBE gel. Pooled duplicates were quantified using PicoGreen and combined by even concentrations. The library was then purified using Ampure XP beads and loaded on to the Illumina MiSeq for sequencing