Minion

WGS data were obtained by Illumina MiSeq and MinIon (Oxford Nanopore Technology, Oxford, UK) and used to confirm PCR results and create the SARS-CoV-2 lineage panel. Libraries for Illumina sequencing were prepared using the Nextera XT library preparation kit (Illumina, San Diego, CA, USA) and then sequenced on a MiSeq instrument (Illumina, San Diego, CA, USA) with a MiSeq Sequence kit v3. FastQC software was used for sequence data quality assessment. Trimmomatic was applied for quality data trimming. Reads were mapped onto reference sequences using BWA. SAMtools-mpileup v1.10.68 [29 (link)] was used to produce draft consensus sequences which were then corrected. Libraries for Oxford Nanopore sequencing were prepared using an SQK-LSK109 DNA Ligation Sequence kit (Oxford Nanopore, Oxford, UK). Sequencing was performed using a MinIon instrument (Oxford Nanopore, Oxford, UK) with a R9.4.1 flowcell. Guppy software was used for base-calling and data quality trimming. Reads were mapped onto the reference sequence Wuhan-Hu-1 SARS-CoV-2 using Minimap2 [30 (link)]. Lineage was defined using PANGO algorithms.

DNA for genome sequencing was extracted and purified using the Qiagen Genomic DNA Preparation Kit (Qiagen) or Maxwell^® DNA Purification Kit (Promega). Illumina short-read libraries were prepared using 100 ng of extracted DNA using the Nextera DNA Library Prep Kit, and paired-end reads were generated using the MiSeq Reagent Kit (v3-600) on MiSeq (Illumina). MinION long-read libraries were prepared from 300 ng of genomic DNA using the Rapid Barcoding kit (SQK-RBK004) and were sequenced using R9 flow cells (FLO-MIN106) on MinION (Oxford Nanopore Technologies) to produce > 100 Mb of data per strain. The Illumina data were preprocessed using Trimmomatic (v0.36) to remove the adapter and low-quality sequences (Bolger et al., 2014 (link)). The quality of the Nanopore sequencing data was assessed using NanoPack (De Coster et al., 2018 (link)) a hybrid assembly with the Illumina and Nanopore reads was performed using Unicycler (v0.4.6) with the default parameters (Wick et al., 2017 (link)). The sequence data have been deposited in NCBI under the accession number, BioProject PRJDB10561.

Whole genome sequence (WGS) data of cfr-positive linezolid-susceptible staphylococci were generated using a combination of Oxford Nanopore MinION (Oxford Nanopore Technologies, Oxford, UK) and Illumina iSeq (Illumina Inc., San Diego, CA, USA). Sequencing data were assembled de novo using Unicycler v.0.5.0. Functional annotation of assembled genome was carried out using the Prokka (v1.14.6) and Rapid Annotation in Subsystem Technology server tool. Integrative data from ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/, accessed on 10 November 2022) of the Center for Genomic Epidemiology and the Comprehensive Antibiotic Resistance Database were used to confirm the presence and location of cfr genes. To analyze the cfr-containing regions, comparative sequence analyses were conducted on the strains sequenced in this study and the previously reported cfr-carrying plasmid pSA12 in a linezolid-resistant ST398 LA-MRSA strain SA12 (GenBank accession no. CP049977) [4 (link)].
The complete genome sequences of four cfr-positive staphylococci were deposited in the NCBI database: S. aureus SA16 strain (GenBank accession no. CP092999-CP093000) and SA19 strain (GenBank accession no. CP110318-CP110319); S. epidermidis SE10 strain (GenBank accession no. CP110320-CP110322); and S. sciuri SSC2 strain (GenBank accession no. CP093001-CP093009), respectively.

Long-read Oxford Nanopore MinION (Oxford Nanopore, UK) and short-read Illumina Hiseq 1500 (Illumina, USA) platforms were combined to generate the whole-genome sequence. To generate short-read sequences, the rapid protocol (2x250 paired-end reads) was performed according to the manufacturer’s protocol for sequencing. For long-read genome sequencing, a MinION sequencing library was prepared using the Nanopore Ligation Sequencing Kit (Oxford Nanopore). The library was sequenced with an R9.4.1 MinION flow cell for a 24 h run using MinKNOW (v2.0) with the default settings. The FAST5 files were base called and converted to FASTQ format in real-time using Guppy (v3.3.0, Oxford Nanopore). Prior to assembly, the raw reads were quality checked with FastQC (v0.11.5, http://www.bioinformatics.babraham.ac.uk/projects/fastqc), and low-quality reads were trimmed using Sickle (v1.33, http://github.com/najoshi/sickle). Long reads were then filtered by quality using Filtlong (v0.2.0) program, available at http://github.com/rrwick/Filtlong.

In order to isolate DNA, down and feather (50 mg) samples were homogenized in 750 µL of Lysis Solution (Zymo Research, Irvine, CA, USA), using a Bead Mill 4 (Thermo Fisher Scientific, Waltham, MA, USA). The DNA was isolated, using ZymoBIOMICS DNA Mini Kit (Zymo Research) according to the manufacturer’s protocol. The isolated DNA was quantified, using a Qubit DNA HS Assay kit (Thermo Fisher Scientific). To isolate DNA from the rinse water, the bacteria in the water was collected using a 0.2 µm filter, and the DNA was isolated from the crashed filter (5 m/s, 300 s, using Beat Mill 4) using the ZymoBIOMICS DNA Mini Kit. 16S rRNA sequencing was performed using an Oxford Nanopore MinION (Oxford Nanopore Technologies, Oxford, UK). Full-length 16S rRNA genes were amplified with 16S_barcode_27f primer (5′-TTT CTG TTG GTG CTG ATA TTG CAG RGT TTG ATC MTG GCT CAG-3′) and 16S_barcode_1492r primer (5′-ACT TGC CTG TCG CTC TAT CTT CGG YTA CCT TGT TAC GAC TT-3′). A DNA library was constructed with PCR Barcoding Kit and Ligation Sequencing Kit (Oxford Nanopore Technologies), according to the manufacturer instruction. The DNA library was applied into MinION, and the output data were analyzed by using EPI2ME software (Oxford Nanopore Technologies).

We sequenced amplicons from 4 UR and 15 INS samples using the Oxford Nanopore MinION (Oxford Nanopore Technologies, Oxford, UK) according to the protocol described in Quick et al. [20 (link)]. Amplicons were barcoded using the Native Barcoding Kit EXP-NBD103 (Oxford Nanopore Technologies, Oxford, UK) and pooled in equimolar fashion. Sequencing libraries were prepared using the 1D Genomic DNA Sequencing kit SQK-LSK108 (Oxford Nanopore Technologies, Oxford, UK). We used AMPure XP beads (Beckman Coulter, Brea, CA, USA) for all purification steps performed as part of library preparation. Prepared libraries were sequenced on R9.4 flowcells (Oxford Nanopore Technologies, Oxford, UK) at the INS in Bogotá and at the Fred Hutchinson Cancer Research Center in Seattle.

To produce long-read sequences of this strain, we applied 400 ng of genomic DNA on the Oxford Nanopore MinION (Oxford Nanopore Technologies, Oxford, UK) device following the Rapid Sequencing protocol (SQK-RAD004). Raw sequence reads were uploaded to the Epi2Me interface (Metrichor, Oxford, UK) for base calling and demultiplexing of MinION data. Epi2Me was used also for examining basic metrics of sequencing abundance and quality. Only base-called data passing Epi2Me quality parameters (q_mean > 6) were downloaded off the cloud in FAST5 and FASTQ formats to use in further analyses. In total, 13,386 MinION reads were obtained with average length and quality score of 6963 bp and 8.3, respectively.