PacBio WGS of Citrobacter freundii
DNA extracts were initially  sheared to an average length of 15kb using g-tubes, as specified by the  manufacturer (Covaris, Woburn, MA, USA). Sheared DNA was used in SMRTbell  library preparation, as recommended by the manufacturer. Quantity and quality  of the SMRTbell libraries were evaluated using the High Sensitivity dsDNA kit  and Qubit fluorometer and DNA 12000 kit on the 2100 Bioanalyzer (Agilent,  Santa Clara, CA, USA). To obtain the longest possible SMRTbell libraries for  sequencing (as recommended by the manufacturer), a further size selection  step was performed using the PippinHT pulsed-field gel electrophoresis system  (Sage Science, Beverley, MA, USA), enriching for the SMRTbell libraries  >15kb for loading onto the instrument. Sequencing primer and P6 polymerase  were annealed and bound to the SMRTbell libraries, and each library was  sequenced using a single SMRT cell on the PacBio RSII sequencing system with  240-minute movies.