Qiamp cador pathogen mini kit extraction

DNA was extracted using the QIAmp Cador Pathogen Mini Kit extraction (Qiagen N.V., Hilden, Germany) according to the manufacturer's instructions. The V3-V4 16S rRNA region was amplified by PCR using the primers reported by Klindworth et al., (2013) [29 (link)] (F-5´ CCTACGGGNGGCWGCAG 3´, R-5´GACTACHVGGGTATCTAATCC 3´). Library preparation was carried out according to the Illumina 16S metagenomic sequencing protocol with minor modifications. Briefly, 16S amplicons were purified with the DNA clean & concentrator kit (Zymo Research, Irvine Cal., USA). Dual indices and Illumina sequencing adapters were attached in a second PCR step using Nextera XT Index Kit V2 (Illumina, San Diego Cal., USA). Finally, amplicons were purified, pooled in equimolar concentrations, and sequenced in a MiSeq Illumina instrument generating paired-end reads of 250 bp.

Respiratory samples for all 127 patients, either nasopharyngeal swabs, oropharyngeal swabs, or tracheal aspirates, were collected and centrifuged for 15 min at 4800×g, and the pellet was used for DNA extraction. DNA was extracted using the QIAmp Cador Pathogen Mini Kit extraction (Qiagen N.V., Hilden, Germany) according to the manufacturer´s instructions. V3–V4 16S rRNA region was amplified by PCR using the primers reported by Klindworth et al.^{54 (link)} (for more information see Supplementary Material S1). Library preparation was done according to the Illumina 16S metagenomic sequencing protocol with few modifications. Briefly, 16S amplicons were purified with the DNA clean & concentrator kit (Zymo Research, Irvine Cal., USA). Dual indices and Illumina sequencing adapters were attached in a second PCR step using Nextera XT Index Kit V2 (Illumina, San Diego Cal., USA). Finally, amplicons were purified, pooled in equimolar concentrations, and sequenced in a MiSeq Illumina instrument generating paired-end reads of 250 bp.