title: 16S rRNA gene sequencing of H. sapiens
design: DNA was extracted from each fecal, meconium, vaginal, breast milk, and oral swab specimen. Samples were thawed at 4C and, in aliquots of 0.15 g per tube, resuspended in 1 ml of 1 X phosphate-buffered saline. Cell lysis was initiated with two enzymatic incubations: 1. using 5 l of lysozyme (10 mg/ml; Amresco, Solon, OH), 13 l of mutanolysin (11.7 U/l; Sigma-Aldrich), and 3 l of lysostaphin (4.5 U/l; Sigma-Aldrich) for an incubation of 30 min at 37C and, 2. using 10 l of proteinase K (20 mg/ml; Research Products International, Mt. Prospect, IL), 50 l of 10% SDS, and 2 l of RNase (10 mg/ml) for an incubation of 45 min at 56C. After the enzyme treatments, cells were disrupted by bead beating in tubes with lysing matrix B (0.1-mm silica spheres; MP Biomedicals, Solon, OH), at 6 m/s at room temperature in a FastPrep-24 (MP Biomedicals). The resulting crude lysate was processed using the ZR fecal DNA miniprep kit (Zymo, Irvine, CA) according to the manufacturers recommendations. The samples were eluted with 100 l of ultrapure water into separate tubes. DNA concentrations in the samples was determined with the Bioanalyzer 2100 DNA 1000 chip (Agilent Santa Clara, CA). The V3-V4 regions of the 16S rRNA gene were amplified by two-step PCR, with amplicon pooling, sequencing on an Illumina HiSeq 2500 instrument.