Ion pgm hi q view kit

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)}.

To profile the bacterial communities, we sequenced the V3 region of the 16S rRNA gene. Firstly, we used the degenerate primer 27FYM (5′-AGR GTT YGA TYM TGG CTC AG-3′) and the primer U534R, targeting the V1–V3 region (500 bp); subsequently, for the semi-nested PCR targeting the V3 region (200 bp), we used the primers B338F_P1-adaptor (B338F 5′-ACTCCTACGGGAGGCAGC-3′) and U534R_A_barcode (U534R 5′-ATTACCGCGGCTGCTGG-3′). Each PCR reaction (sample) contained a unique IonXpress Barcode Adapter attached to the reverse primer. No-template controls were processed with the clinical samples. The template preparation was performed using the Ion PGM Hi-Q View kit on the Ion OneTouch™ 2 System (Life Technologies, Grand Island, New York, NY, USA) and was sequenced using the Ion PGM Hi-Q View sequencing kit (Life Technologies, New York, NY, USA) with the Ion PGM™ System technology.
The FastQ files were processed using QIIME 2.0, version 2022.2, retaining reads with Q ≥ 20 and a read length 180 bp, after DADA2 denoising. For the taxonomy assignment, Silva v138 was used, with a BLAST+ consensus. Further analysis was carried out on a random subset of 10,000 reads/sample, using a similarity threshold of 97%.

DNA was extracted from the swabs using the NucliSENS^® easyMAG^® system (BioMèrieux, Gorman, North Carolina, CA, USA), following the manufacturer’s instructions, starting from 500 µL of sample and with an elution volume of 50 µL. All DNA samples were stored at −80 °C prior to further processing.
Firstly, a 500 base pair region of the V1-V3 portion of the 16S rRNA gene was amplified and subsequently the 200 base pair region of the V3 portion was amplified as well, as described elsewhere [12 (link)]. The V3 amplicon was used for template preparation by the Ion PGM Hi-Q View kit on the Ion OneTouch™ 2 System (Life Technologies, Gran Island, New York, NY, USA) and sequenced using the Ion PGM Hi-Q View sequencing kit (Life Technologies, New York, NY, USA) with the Ion PGM™ System technology. Negative controls, including a no template control, were processed with the clinical samples.