Ion pgm hi q view sequencing kit

The V2–V3 portion of the 16S 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, Wilmington, MA, United States) 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, CA, United States). 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, Norcoss, GA, United States), 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 with the clinical samples (Campisciano et al., 2017 (link)).

Eighty‐four samples already available in our laboratories were selected to perform the ddRAD‐seq genotyping experiment. These samples belonged to individuals collected across most of the contemporary geographical range of the species and covering all the previously known genetic clusters. Of these, 70 were retained for further analyses after quality control and filtering (see next paragraph and Figures S1–S12).
DNeasy Blood and Tissue Kit (Qiagen) and Quick‐DNA Universal Kit (Zymo Research) were used to extract genomic DNA from FTA cards and whole blood stored at −20°C, respectively, following manufacturer instructions. DNA was quantified with Qubit using the dsDNA BR kit (Invitrogen). ddRAD‐seq libraries were prepared using SbfI and NcoI for restriction digestion. Sequencing was performed on the Ion Torrent PGM with the Ion PGM Hi‐Q View Sequencing kit (Life Technologies) following manufacturer's instructions. See Supplementary Methods for more details about library preparation and sequencing.

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)}.

Genomic DNA from confirmed L. monocytogenes isolates was purified using the Gentra Puregene Yeast/Bacterial Kit (Qiagen, Hilden, Germany). DNA was quantified by Qubit 3.0 (Life Technologies, Carlsbad, CA, USA) and stored at −20 °C until library preparation. DNA libraries were prepared using the Ion Xpress Plus Fragment Library kit using the enzymatic fragmentation procedure following the manufacturer’s instructions (Life Technologies, ThermoFisher Scientific, Carlsbad, CA, USA). Each library was indexed using Ion Xpress™ Barcode Adapters (Life Technologies, ThermoFisher Scientific). Ion Library TaqMan Quantitation Assay for qPCR (7500 Fast, Life Technologies, ThermoFisher Scientific) was used for library quantification. Template preparation was performed using the Ion OneTouch™ 2 System and the Ion PGM™ Hi-Q™ View OT2 400 Kit (Life Technologies, ThermoFisher Scientific). The libraries were sequenced with the Ion Torrent PGM sequencer (Life Technologies, ThermoFisher Scientific) using the Ion PGM™ Hi-Q™ View Sequencing Kit (Life Technologies, ThermoFisher Scientific), following the manufacturer’s protocols. To obtain deep sequencing results for each sample, seven barcoded samples were sequenced in each Ion chip 318 (Life Technologies, ThermoFisher Scientific) with more than 40× coverage per sample.

Peptide-encoding region of bacteriophage genome was amplified by PCR using Phusion Green Hot Start II High-Fidelity DNA Polymerase (Thermo Scientific, F537L) (reaction volume: 25 μl, list of primers added as Supplementary Table S1). Cycling conditions: denaturation at 98°C for 30 s, followed by 25 amplification cycles (10 s at 98°C, 21 s at 72°C), and final elongation (72°C for 5 min). Polymerase chain reaction (PCR) products were purified using AMPure XP Bead Based Next-Generation Sequencing Cleanup system (Beckmann Coulter, A63881) using 1.8 μl of beads per 1 μl of PCR products. Purified PCR products were quantified using Agilent Bioanalyzer 2100 Instrument using the High sensitivity DNA Kit (Agilent, 5067-4626). Ion Torrent Emulsion PCR and enrichment steps were performed using Ion PGM HiQ View OT2 kit (Life technologies, A29900). HTS was performed using Ion Torrent™ Personal Genome Machine™ (ION-PGM) using Ion PGM HiQ View sequencing kit (Life technologies, A30044) and Ion 316v2 chips (Life Technologies, 448149). The FASTQ sequence files were converted to text files and translated using in-house developed python scripts.

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%.