16S rRNA Amplicon Sequencing Protocol

The V2–V3 portion of the 16 S rRNA was amplified, using the primer set F101- R534, with a different IonXpress barcode per sample attached to the reverse primer. PCR reactions were performed using the Kapa Library Amplification Kit (Kapa Biosystems, Massachusetts, USA) and BSA 400 ng/µL, under the following conditions: 5 min at 95 °C, 30 s at 95 °C, 30 s at 59 °C, 45 s at 72 °C, and a final elongation step at 72 °C for 10 min. DNA after normalization was quantified with a Qubit® 2.0 Fluorometer (Invitrogen, Carlsbad, California, USA). The amplicon size was checked on a 2% agarose gel. The subsequent step of PCR purification was carried out using the Mag-Bind® Total Pure NGS beads (OMEGA Bio-Tek, Georgia, USA), retaining fragments >100 bp. Template preparation was performed by the Ion PGM Hi-Q View kit on the Ion OneTouch^TM 2 System (Life Technologies, Grand Island, NY, United States) and sequenced using the Ion PGM Hi-Q View sequencing kit (Life Technologies, Grand Island, NY, United States) with the Ion PGM^TM System technology. Negative controls, including a no-template control, were processed along with samples as previously suggested^{74 (link)}.