16S amplicon sequencing of human sputa
Sputum samples were processed with the MagNa Pure 203 System and MagNa Pure LC DNA Isolation kit (Roche), according to the protocols provided by the manufacturer. PCR amplification of the V1-V2 region (349 bp) of the 16S RNA gene was carried out using primers 5-AGR GTT YGA TYM TGG CTC AG-3 and 5-TGC TGC CTC CCG TAG GAG T-3, where R could be A or G, Y could be C or T, M could be A or C. Platinum PCR Super Mix High Fidelity (Life Technologies) was used, according to the manufacturers recommendations, with a no template sample as a negative control. PCR products were purified by MinElute PCR Purification Kit (Qiagen) and quantified using Qubit (Life Technologies). Sequencing libraries were prepared with the TruSeq ChIP Sample Prep Kit (Illumina) starting from 10 ng of PCR-amplified DNA and checked by Qubit and Fragment Analyzer before loading the sequencing cell. Multiplexed high-throughput sequencing was performed on a MiSeq instrument (Illumina), with MiSeq Reagent kit V2 500 cycles (Illumina), where libraries were diluted to 8 pM and pooled according to the origin of the sputum samples. PhiX (PhiX Control V110, Illumina) was spiked in to generate diversity in the clusters.