Genome Assembly and Vancomycin Resistance Analysis

Raw FASTQ sequencing reads from the Illumina MiSeq whole-genome sequencing were processed using an assembly pipeline generated with the SeqSphere+ version 6 (Ridom GmbH, Münster, Germany)⁴. FastQC (Andrews et al., 2010 ) was used to perform a quality check of the read files to assess the sequencing quality scores, total number of reads, and GC content. For the removal of the Nextera XT index library adapters, Trimmomatic (Bolger et al., 2014 (link)) was applied to achieve an average Q score of 30 in a sweeping window of 20 bases. The BWA plug-in in the SeqSphere+ software was used for the assembly of genome sequences of each isolate by mapping the paired-end reads to the complete reference genome of E. faecium DO (TX16_NC-017960) (Qin et al., 2012 (link)). The resultant contiguous consensus sequences (contigs) were exported for in silico identification of vancomycin-resistance (van) locus using the ResFinder server on the Centre for Genomic Epidemiology (CGE) online tool⁵. The settings used were a minimum sequence identity threshold of 90% and a genome length identity cut-off of 60%. The assembled genome sequences were queried against the MLST tool⁶ from the CGE database to determine the sequence type of the isolates. The E. faecium MLST database⁷ was also queried to confirm the sequence types.