16S sequencing of human lung microbiome
DNA was extracted with the DNeasy PowerLyzer PowerSoil DNA Isolation Kit (QIAGEN, Valencia, CA), following the manufacturer's instructions. PCR amplification was performed on the 16S rRNA gene V4 hypervariable region using the 515F and 806R primers, with a 12-bp unique Golay barcoding [18]. PCR reactions were performed with an initial denaturation of 95 C for 5 min, followed by 15 cycles of 95 C for 1 min, 55 C for 1 min, and 68 C for 1 min, followed by 15 cycles of 95 C for 1 min, 60 C for 1 min, and 68 C for 1 min, and a final extension for 10 min at 68 C on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). The PCR products were purified using a QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA) and quantified using a Qubit 2.0 Fluorometric High Sensitivity dsDNA Assay (Life Technologies, Carlsbad, CA). PCR products were pooled relative to their band intensity. KAPA LTP Library Preparation Kit (KAPA Biosystems, Wilmington, MA) was used on the combined purified PCR products according to the manufacturer's protocol and the size integrity of the amplicons with Illumina indices was validated with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) at the Genomics Core at Albert Einstein College of Medicine. High-throughput amplicon sequencing was conducted on a HiSeq (Illumina, San Diego, CA) using 2  150 paired-end fragments.