@misc{raven_leek_blane_girgis_akram_brown_peacock_2021, 
  title={Evaluation of methods for whole genome sequencing of Enterococcus faecium in a diagnostic laboratory}, 
  DOI={10.1101/2021.11.09.21265457}, 
  abstractNote={<jats:p><jats:italic>Enterococcus faecium</jats:italic> is an important nosocomial pathogen associated with hospital transmission and outbreaks. Based on growing evidence that bacterial whole genome sequencing enhances hospital outbreak investigation of other bacterial species, our aim was to develop and evaluate methods for low volume clinical sequencing of <jats:italic>E. faecium</jats:italic>. Using a test panel of 22 <jats:italic>E. faecium</jats:italic> isolates associated previously with hospital transmission, we developed laboratory protocols for DNA extraction and library preparation, which in combination with the Illumina MiniSeq can generate sequence data within 24 hours. The final laboratory protocol took 3.5 hours and showed 98% reproducibility in producing sufficient DNA for sequencing. Repeatability and reproducibility assays based on the laboratory protocol and sequencing demonstrated 100% accuracy in assigning species, sequence type (ST) and (when present) detecting <jats:italic>vanA</jats:italic> or <jats:italic>vanB</jats:italic>, with all isolates passing the quality control metrics. Minor variation was detected in base calling of the same isolate genome when tested repeatedly due to variations in mapping and base calling, but application of a SNP cut-off (&lt;15 SNPs) to assign isolates to outbreak clusters showed 100% reproducibility. An evaluation of contamination showed that controls and test <jats:italic>E. faecium</jats:italic> sequence files contained &lt;0.34% and &lt;2.12% of fragments matching another species, respectively. Deliberate contamination experiments confirmed that this was insufficient to impact on data interpretation. Further work is required to develop informatic tools prior to implementation into clinical practice.</jats:p>}, 
  publisher={Cold Spring Harbor Laboratory}, 
  author={Raven, Kathy E and Leek, Danielle and Blane, Beth and Girgis, Sophia and Akram, Asha and Brown, Nicholas and Peacock, Sharon}, 
  year={2021}, 
  month={Nov}
  }