Genomic Analysis of VREfm Isolates Using Illumina MiSeq

The VREfm isolates were sequenced on the Illumina MiSeq platform (Illumina Inc., San Diego, USA) as previously described.^{7 (link)} Raw reads were trimmed using BBDuk (https://sourceforge.net/projects/bbmap/) with the parameters ktrim = r, k = 23, mink = 11, hdist = 1, tbo, qtrim = r and minlength = 30, and assembled with SKESA v.2.2 with default settings except inclusion of the parameter –allow_snps. Only assemblies with a genome size in the range 2.7–3.2 Mb, a minimum depth of coverage of 30, and N50 of minimum 10 000 were included in the study. We used seqsphere+ v.8.2.0 (Ridom GmbH, Munster, Germany; seqsphere/">http://www.ridom.de/seqsphere/) with the previously published scheme for E. faecium for the initial bioinformatic analysis.^{8 (link)} Sequencing reads were aligned, and SNPs were called using the NASP pipeline v.1.1.2.^{9 (link)} In the pipeline, duplicated regions in the reference were masked using NUCmer v.3.1, reads were subsequently mapped to the E. faecium Aus0004 (CP003351.1) reference genome with BWA-mem v.0.7.16 and SNPs were called with GATK v.3.8.0.^{10–13 (link)} Consensus bases had a minimum coverage of 10 and a minimum proportion of 0.9 for the called base. Pairwise comparisons between isolates of patients were performed using the consensus base matrix. Recombination regions were detected for every clone group and filtered out using Gubbins v.3.2.1.^{14 (link)}