There is growing evidence for the value of bacterial whole genome sequencing in hospital outbreak investigation. Our aim was to develop methods that support efficient and accurate low throughput clinical sequencing of methicillin-resistant Staphylococcus aureus (MRSA). Using a test panel of 25 MRSA isolates associated previously with outbreak investigations, we devised modifications to library preparation that reduced processing time by 1 hour. We determined the maximum number of isolates that could be sequenced per run using an Illumina MiniSeq and a 13 hour (overnight) run time, which equated to 21 MRSA isolates and 3 controls (no template, positive and negative). Repeatability and reproducibility assays based on this sequencing methodology demonstrated 100% accuracy in assigning species and sequence type (ST) and detecting mecA. Established genetic relatedness between isolates was recapitulated. Quality control (QC) metrics were evaluated over nine sequencing runs. 168/173 (97%) test panel MRSA genomes passed QC metrics based on the correct species assigned, detection of mecAand ST, and depth/coverage metrics. An evaluation of contamination in these 9 runs showed that positive and negative controls and test MRSA sequence files contained <0.14% and <0.48% of fragments matching another species, respectively. Deliberate contamination experiments confirmed that this was insufficient to impact on data interpretation. These methods support reliable and reproducible clinical MRSA sequencing with a turnaround time (from DNA extraction to availability of data files) of 24 hours.

Credit:  Kathy E. RavenBeth BlaneDanielle LeekCarol ChurcherPaula Kokko-GonzalesDhamayanthi PugazhendhiLouise FraserJason BetleyJulian ParkhillSharon J. Peacock

Credit CDC

DOI: 10.1128/JCM.00180-19