Bacterial Genomic DNA Isolation and Sequencing

Genomic DNA was isolated from overnight bacterial cultures of the strains using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA), with the enzyme pretreatment step modified to 50 μg/mL lysostaphin and 500 μg/mL lysozyme in 10 mM Tris-HCl 1 mM EDTA (pH 8.0). Sequencing libraries were prepared using the TruSeq DNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. WGS was performed using the Illumina MiSeq platform (Illumina). The paired-end sequencing reads were assembled using CLC Genomics Workbench 7.3 (CLC Bio, Aarhus, Denmark). Genes were predicted using Glimmer 3 [15 (link)] and annotated by homology searches against the Clusters of Orthologous Groups and SEED databases (https://theseed.org) [16 (link)]. To validate the results, the assembled sequences were compared with the N315 and Mu50 MRSA reference genomes.