16S rRNA gene sequencing of H. sapiens
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.