Microbiomes of bronchial lavage fluids of humans

In this study, 16S sequencing was performed to assess the bacterial microbiomes in DNA isolated from bronchial lavage fluid samples of human subjects with or without cancer. • Controls: eight neg. ctrl. for BAL procedure (BAL buffer only put through BAL instrument without patient), three neg. ctrl. for DNA extraction (empty tube), and two neg. ctrl. for 16S PCR (no template); four pos. ctrl. (ZymoBIOMICS™ Microbial Community DNA Standard, Zymo® cat. #6305). • DNA isolation & quantification: ZymoBIOMICS DNA Microprep Kit D4301, with 20 ul elution; DNA quantified by Thermo® Qubit DNA Kit. • 16S PCR and library preparation: As per Illumina® Metagenomics Guide, using the guide's suggested V3-V4 primers; Kapa Biosystems® KAPA HiFi ReadyMix for PCR, with 25 cycles at annealing temperature of 55 ºC and 25 ng DNA input (when available; otherwise 19.2 ul DNA sample). An ~464 bp amplicon of bacterial 16S rRNA V3-V4 region was amplified with forward and reverse primers of sequences TCGTCGGCAGCGTC-ad-CCTACGGGNGGCWGCAG and GTCTCGTGGGCTCGG-ad-GACTACHVGGGTATCTAATCC, respectively (ad = AGATGTGTATAAGAGACAG). • Amplified DNA was purified with AMPure XP beads and was indexed with Nextera XT index kit in an 8-cycle PCR to generate sequencing libraries. Libraries were purified with AMPure XP beads and 12 pmoles of each library was sequenced on a MiSeq instrument (Illumina) with v3 sequencing reagents to obtained paired 300 bp reads. A PhiX library was spiked in at 10% molarity. Multiplexed libraries were sequenced thrice using three MiSeq sequencing runs. Sequencing data uploaded to ENA has merged raw (CASAVA de-multiplexed) fastq.gz files of the three runs.