67 Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC Veterinary Institute part of Wageningen UR, Lelystad, The Netherlands), were asked to submit pampC confirmed E. coli strains to be evaluated in our algorithm. DNA isolation, library preparation, and DNA sequencing of training set and validation set 2 Bacterial DNA was extracted by a CTAB-based method. Colonies were resuspended in 400 µL TE buffer (10 mM Tris pH8.0, 1 mM EDTA) and 50 µL of 10 mg/mL lysozyme was added. Samples were incubated for 60 min at 37oC, where after 75 µL of 0.7 mg/ mL proteinase K in 10% SDS was added followed by an incubation for 10 min at 65oC. The samples were mixed with 100 µL of 5M NaCl and 100 µL CTAB/NaCl solution (1% N-cetyl-N,N,N,-trimethyl ammonium bromide in 0.7 M NaCl) and incubated for 10 min at 65oC. DNA was further isolated using chloroform/isoamylalcohol extraction. DNA was precipitated from the aqueous phase by adding an equal amount of 2-propanol and subsequent incubation for 20 min at -20oC. Samples were centrifuged for 10 min at 11.000g. DNA pellets were washed with 1 mL cold 75% ethanol and centrifuged for 5 min at 11.000g. DNA pellets were air-dried for 15 min at room temperature and dissolved in 100 µL TE buffer. DNA samples were quantified using the QuantiFluor dsDNA system (Promega, Madison, WI, USA). A fragmented genomic DNA library was prepared using a NexteraXT DNA sample preparation kit (Illumina, San Diego, CA, USA). Library purification was performed using Agencourt AMPure XP beads (Beckman Coulter, Bread, CA, USA). Subsequent sequencing was conducted in a paired-end 2 x 150 bp mode using an Illumina NextSeq500 sequencer (Illumina, San Diego, CA, USA). DNA isolation, library preparation, and DNA sequencing of validation set 1 Colonies were suspended in 1000 µL TE buffer. Bacterial DNA was extracted using MagNA Pure LC Total Nucleic Acid Kit - High Performance on a MagNA Pure LC instrument (Roche Diagnostics International Ltd, Rotkreuz, Switzerland) according to the manufacturer’s Protocol. A total of 200 µL bacterial solution was used for DNA extraction, together with Lysis/Binding buffer and proteinase K according to kit-protocol, with an elution volume of 100 µL. DNA concentration was normalized to 0.2 ng/µl by adding TE buffer to eluate. DNA samples were quantified using the Qubit Fluorometer dsDNA 3.0 system (Thermofisher Scientific, Waltham, MA, USA). A fragmented genomic DNA library was prepared using a NexteraXT DNA sample preparation kit. Library purification was performed using Agencourt AMPure XP beads. Subsequent sequencing was conducted in a paired-end 2 x 300 bp mode using an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA). 4
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