16S rRNA Gene Amplification and Sequencing

PCR amplification of two variable (V1-V2) regions (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’ (Microsynth AG, Switzerland), where “R” could be A or G, “Y” could be C or T, “M” could be A or C [30 (link)]. Platinum PCR Super Mix High Fidelity (Life Technologies) was used, according to the manufacturer’s 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 (Agilent) before loading the sequencing cell. Multiplexed (S1 Table) high-throughput sequencing was performed at the Ecole Polytechnique Fédérale de Lausanne 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 V3, Illumina) was spiked in (15%) to generate diversity in the sequencing clusters.