R9 flow cell

All phage genomic DNA was extracted using the Norgen phage DNA isolation kit (Norgen Biotak, Birmingham, UK), following the manufacturer’s instructions. Each phage DNA library was constructed using the QIA seq FX DNA library kit (Qiagen), and sequencing was performed on the Illumina MiSeq platform. Genome assembly was performed using Shovill with default settings. Phage contigs were filtered based on a contig length <200 and coverage of <25. Bacteria and phage strains were annotated using prokka (57 (link)) or PGAP (58 (link)) version 2021-07-01.build5508. For Nanopore long-read sequencing, we used the Monarch HMW DNA Extraction Kit for Tissue (NEB, MA, USA) following the manufacturer’s instructions.
A long-read library was prepared using the Rapid Barcoding kit (Oxford Nanopore Technologies, Oxford, United Kingdom, catalog number: SQK-RBK004) and sequenced on an R9 flow cell (Oxford Nanopore Technologies, catalog number: FLO-MIN106) using a GridION device (Oxford Nanopore Technologies). Basecalling was performed using Guppy version 5.0.12 in high accuracy mode. The obtained long reads, as well as the MiSeq short reads, which were trimmed using fastp v0.20.1, were assembled using Unicycler v0.4.8 with default parameters. Annotation was conducted using PGAP version 2021-07-01.build5508.

Leftover SARS-CoV-2 PCR-positive respiratory specimens were used for virus sequencing. Samples resulting in a C_T value of ≤27 were selected for whole-genome sequencing, done by modifying a previously reported protocol (52 (link)). Briefly, reverse transcription was performed using the LunaScript RT supermix kit (New England Biolabs, Ipswich, MA, USA). Multiplex PCR for library preparation was performed using the SARS-CoV-2 ARTIC Network V2/V3 amplicon set. Amplicon pools were quantified using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Prepared DNA was loaded onto an R9 flow cell (Oxford Nanopore Technologies, Oxford, UK) and run in a MinION sequencer (Oxford Nanopore Technologies, Oxford, UK). Bioinformatics analysis of raw sequencing data was performed following a protocol described elsewhere (53 ). Additionally, sequences were analyzed using the bioinformatics tools Pangolin (54 (link)), Nextclade (55 (link)), and Nextstrain (56 (link)). Seven specimens did not yield full genomes, and therefore, a total of 881 full genomes were characterized.

Library preparation was performed using the Rapid Barcoding Kit (SQK-RBK004) provided by Oxford Nanopore Technologies with an R9 flow cell (FLO-MIN106). The procedure took approximately 1 h per 12 samples. MinION libraries were created with 400 ng of total genomic DNA for each sample with an adjusted volume of 7.5 μL Nuclease-free water. A clean-up step using Agencourt AMPure XP beads (Beckman coulter, Munich, Germany) at 1X concentration was used to discard short fragments as recommended by the manufacturer. DNA quantity was assessed using Qubit fluorimeter. Promega calculator (https://www.promega.es/resources/tools/biomath/) was used to convert μg dsDNA into pmol. This parameter must fall between 0.2–0.25 pmol, as specified in the protocol kit. This amount refers to the prepared library that must be loaded into the MinION flow cell. Once the library has been loaded, we initiated a standard 18 h sequencing procedure using the MinKNOW software, and by using the live basecalling feature.