*4.2. Genomic DNA Preparation, High-Throughput Sequencing, Assembly, and Annotation*

The *Enterococcus* spp. strains were grown in BHI at 37 ◦C for 18 h. Genomic DNA was extracted using a commercial kit (QIAGEN DNeasy Blood & Tissue Kit, San Luis, MO, USA). Manufacturer instructions were followed with minor modification, namely, the addition of 50 μL of lysozyme (50 mg/mL) and 10 μL mutanolysin (2500 U/mL, Sigma-Aldrich, Germantown, MD, USA) for 30 min at 37 ◦C before the addition of 20 μL proteinase K (20 mg/mL). Extracted DNA was quantified using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Life Technologies, Carlsbad, CA, USA). Libraries for genome sequencing were prepared using the Nextera XT DNA kit and index primers

(Illumina), and reads were generated by HiSeq/MiSeq reagent kit version 2 with 250 cycles on an Illumina HiSeq/Miseq platforms. Reads were subjected to de novo assembly using the CLC genomics workbench v8.0.3, and open reading frames (ORFs) were predicted using the NCBI Prokaryotic Annotation Pipeline—PGAP [100]. The enterococci species assignment was confirmed by pairwise comparison of their average nucleotide identity (ANI) using JSpeciesWS [101] and the following reference genomes available from GenBank (https://www.ncbi.nlm.nih.gov (accessed on 15 December 2020): *Enterococcus avium* ATCC 14025; *Enterococcus casseliflavus* ATCC 12755; *Enterococcus faecalis* ATCC 19433; *Enterococcus faecium* Aus0004 (Clade A1); *Enterococcus faecium* EnGen0007 (Clade A2); *Enterococcus faecium* Com12 (Clade B); *Enterococcus hirae* ATCC 9790; *Enterococcus lactis* KCTC 21015; *Enterococcus mundtii* ATCC 882. The GenBank accession number of reference strains is presented in Supplementary Table S2.
