Fecal microbiome 16S rRNA sequencing

Total DNA was extracted by bead beating from approximately 50 mg of each fecal sample using the MOBIO PowerFecal DNA Isolation Kit (MOBIO Laboratories, Carlsbad, CA, USA) according to the manufacturer's protocol. DNA isolated from fecal samples was quantified using a NanoDrop (ThermoScienctific), and the V5–V6 regions of the 16S rRNA gene were PCR amplified using Accura High Fidelity Polymerase, with the addition of barcodes for multiplexing. The forward and reverse primers were the V5F and V6R sets [68 (link)], chosen in part to allow dual coverage of the entire region. The barcoded amplicons were pooled and Illumina adapters were ligated to the reads. A single lane on an Illumina MiSeq instrument was used (250 cycles, 300 bp, paired-end) to generate 16S rRNA gene sequences yielding 175,784 Pass Filter (PF) reads per fecal sample (SD = 72,822) and ~12.65 million total PF reads (4.9Gb of data). Raw sequencing data (fastq files) are available through MG-RAST [Project ID: mgp15238].