Vancomycin resistant Enterococcus VRE_2
DNA library preparation and sequencing was performed according to the manufacturer’s  instructions and reflects the P5-C3 sequencing enzyme and chemistry, respectively. In  short, 5 μg of extracted, high-quality, genomic DNA from each of twelve VRE isolates  was verified using Qubit analysis to quantify the mass of double-stranded DNA present.  After quantification, each sample was diluted to 150 μL using Qiagen elution buffer at 33  μg / μL. The 150 μL aliquots were individually pipetted into the top chambers of Covaris  G-tube spin columns and sheared gently for 60 seconds at 4500 rpm using an  Eppendorf 5424 benchtop centrifuge and repeated at 60 seconds at 4500 rpm to further  shear the DNA and place the aliquot back into the upper chamber, resulting in a  ~20,000 bp DNA shear, verified using a DNA 12000 Agilent Bioanalyzer gel chip. The  sheared DNA isolates were then re-purified using a 0.45X AMPure XP purification step.  There was no evidence of plasmids below 10kb, thus only one large insert, sizeselected  library was constructed.  After purification, 2.1 to 3.4 μg of each purified and sheared sample was taken into DNA  damage and end-repair. Briefly, the DNA fragments were repaired using DNA Damage  Repair solution (1X DNA Damage Repair Buffer, 1X NAD+, 1 mM ATP high, 0.1 mM  dNTP, and 1X DNA Damage Repair Mix) with a volume of 21.1 μL and incubated at  37ºC for 20 minutes. DNA ends were repaired next by adding 1X End Repair Mix to the  solution, which was incubated at 25ºC for 5 minutes followed by the second 0.45X  Ampure XP purification step. Next, 0.75 μM of Blunt Adapter was added to the DNA  followed by 1X template Prep Buffer, 0.05 mM ATP low and 0.75 U/μL T4 ligase to  ligate (final volume of 47.5 μL) the SMRTbell adapters to the DNA fragments. This  solution was incubated at 25ºC overnight followed by a 65ºC 10-minute ligase  denaturation step. After ligation, the library was treated with an exonuclease cocktail to  remove un-ligated DNA fragments using a solution of 1.81 U/μL Exo III 18 and 0.18  U/μL Exo VII, then incubated at 37ºC for 1 hour. Two additional 0.45X Ampure XP  purifications steps were performed to remove <2000 bp molecular weight DNA and  organic contaminant.  Upon completion of library construction, samples were validated as ~20 kb using  another Agilent DNA 12000 gel chip. All libraries were sufficient for additional size  selection to remove any library molecules <7,000 bp. This step was conducted using  Sage Science Blue Pippin 0.75% agarose cassettes to select library in the range of  7,000-50,000 bp. 11% to 27% of the input library eluted from the agarose cassette and  was available for sequencing. This yield was sufficient to proceed to primer annealing  and DNA sequencing on the PacBio RSII machine. Size-selection was confirmed by  Bio-Analysis and the mass was quantified using the aforementioned Qubit assay.  Primer was then annealed to the size-selected SMRTbell with the full-length libraries  (80ºC for 2 minute 30 seconds followed by decreasing the temperature by 0.1º to 25Cº).  The polymerase-template complex was then bound to the P4 enzyme using a ratio of  10:1 polymerase to SMRTbell at 0.5 nM for 4 hours at 30ºC and then held at 4ºC until  ready for magbead loading, prior to sequencing. The magnetic bead-loading step was  conducted at 4ºC for 60-minutes per manufacturer’s guidelines. The magbead-loaded,  polymerase-bound, SMRTbell libraries were placed onto the RSII machine at a  sequencing concentration of 50 pM and configured for a 180-minute continuous  sequencing run. Sequencing was conducted to ample coverage across four SMRTcells  for each isolate. Data was then generated and assembled using the HGAP3  SMRTportal assembly pipeline, filtering at 0.80 RQ, 500 bp sub-readlength, and  standard pre-assembly pipeline parameters