human nasopharyngeal microbiome
DNA was extracted from nasopharyngeal swab samples using the ZymoBIOMICSTM 96 MagBead DNA Kit (Zymo Research) following manufacturers instructions. The extraction tubes were agitated using Tissue lyser II (Qiagen, Hilden, Germany) at 30 Hz/s for 10 min. Mock community DNA was included as positive control for library preparation (Zymobiomics Microbial Community DNA, ZymoResearch, Irvine, CA, USA). After DNA extraction, the V3-V4 region of the 16S rRNA of bacteria was amplified using regions specific primers (For: 5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3, Reverse 5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3). The PCR was performed in 10 l final volume with 0.2-M primer concentration. The PCR cycle included:  3 min at 95 C (initial denaturation) followed by 35 cycles; 30 s at 95 C 30s at 55 C, and 30s at 72 C, and a final elongation step of 5 min at 72 C. PCR products were purified using AMPure XP beads (Beckman Coulter, Nyon, Switzerland) with a 0.9x ratio according to manufacturers instructions. Nextera barcoded adapters were also included as part of the library preparation protocol (using Nextera XT v2 adaptor primers) resulting in 450 bp insert sizes. 25 l of the second PCR product were purified with SequalPrep normalization kit (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer's protocol. The final library pool was quantified by qPCR using Kapa library quantification kit for Illumina Platforms (Kapa Biosystems, SigmaAldrich, Saint Louis, MO, USA) on an ABI 7900HT real-time cycler (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA). Sequencing was performed using Illumina MiSeq (2300 bp).
A0000748