16S rRNA-Seq of the upper airway microbiome:  Nasal brushings (nasopharynx and nasal vestibule) from individuals ages 16 to 78 : Sample NKRMzgsf
We froze the nasal brushes at 20C immediately after collection, shipped them on dry ice, and stored them at -80C. We used the BiOstic Bacteremia DNA Isolation Kit (1224050) for DNA extraction and the Nanodrop 1000 spectrophotometer (Thermo Scientific, IL, USA) to assess DNA concentration and purity.  We used 62 different region-specific primers labelled with a unique 12-base Golay barcode sequence in the reverse primer to amplify the 16S rRNA gene V4 region following conditions in the Caporaso et al. protocol [1].  We used the Agilent Bioanalyzer DNA 1000 (Agilent Technologies, CA, USA) to conduct quality control in the final libraries prior to pooling. We pooled the libraries into 8 pools of 62 samples and sequenced them on the HiSeq2000 platform (Illumina Inc., CA, USA) under a single end 102 base-pair protocol.  1. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 2012;6:16214.