Gut Microbiome Profiling from Fecal, Ileal, and Cecal Samples

DNA was extracted from fecal, ileal, and cecal samples using the Mobio 96-well extraction kit following the manufacturer’s instructions. For amplicon library generation, the V4 region of the 16S rRNA gene was amplified with gene-specific primers, as described^{38 (link)}. The reverse amplification primers contained a 12-base pair Golay barcode for multiplexed sequencing runs that support pooling up to 864 samples. Amplicons were prepared in triplicate, pooled, and quantified. The 254 bp V4 region was sequenced using the Ilumina MiSeq 2 × 150 bp platform. OTUs were picked using GreenGenes 13_8 for reference, using the open reference picking strategy.