Amplification and sequencing
The microbiota of the 61 BAL samples included in the study was characterized by high throughput sequencing of the 16S rRNA gene. For this purpose, the 16S Ion Metagenomics kit (Life Technologies) was used, following the manufacturer’s instructions. Briefly, this method includes two separate PCR reactions, amplifying, respectively, V2-4–8 and V3-6, V7-9 regions. Each PCR reaction was carried out on 5 ng of microbial DNA. The thermal profile for both PCR reactions consisted of 1 cycle at 95 °C for 10 min, 30 cycles consisting of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 20 s, and a final extension cycle at 72 °C for 7 min. After amplification, PCR products were purified using the Agencourt AMPure beads (Beckman Coulter Inc, Atlanta, Georgia), eluted in Low TE buffer and quantified by the Qubit dsDNA HS Assay kit (Life Technologies). After amplification, a pool of 100 ng of total DNA for each sample (50 ng from each reaction) was used for library preparation. Libraries were barcoded using Ion Xpress Barcodes Adapters (Life Technologies) and amplified in an emulsion PCR on the Ion Chef system (Life Technologies) according to the manufacturer’s instructions. Sequencing was performed on the Ion S5 System (ThermoFisher) using the Ion 330 Chip kit (Life Technologies). The raw sequences were submitted to NCBI under BioProject accession number PRJNA892871.