WGS of Burkholderia pseudomallei isolate: environmental soil sample
uspected B. pseudomallei colonies were inoculated into 5 ml liquid tryptic soy broth (TSB) and incubated at 37C overnight while shaking.  The following day, 1 ml was withdrawn, the cells were pelleted, and genomic DNA was isolated using the MasterPure Complete DNA and RNA Purification Kit (LGC Biosearch Technologies; Middleton, WI) according to the manufacturers instructions.  Quantitation and Quality Control of Genomic DNA.  Purified genomic DNA was quantified using Qubit dsDNA BR assays (ThermoFisher Scientific; Waltham, MA) and a Qubit 4 fluorimeter.  DNA integrity was evaluated by electrophoresis using Genomic DNA ScreenTape, genomic reagents and a TapeStation 4150 (Agilent Technologies; Santa Clara, CA). Short-reads shotgun libraries were prepared using NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs; Ipswich, MA) following the manufacturers instructions. Briefly, 26 l of DNA at 2-4 ng/l was first fragmented enzymatically via 20 min incubation at 37C.next, hairpin sequencing adaptors, containing 5-dT overhangs and a U ribonucleotide in the hairpin loop, were added to the fragmented DNA by ligation, and subsequently cleaved at the U sites.  The libraries were then amplified and indexed by PCR using NEBNext Unique Dual Indexes.  Prior to sequencing, the libraries were evaluated for quality using Agilent D1000 kit (Agilent Technologies; Santa Clara, CA).  The libraries that passed quality control were then quantified using Qubit dsDNA BR assay (ThermoFisher Scientific; Waltham, MA), pooled, and sequenced using a NovaSeq 6000 S4 Reagent Kit,v1.5 300 cycles, and a NovaSeq 6000 sequencer (Illumina; San Diego, CA).  Long-reads libraries were prepared using Ligation kits with native barcodes (Oxford Nanopore Technologies, Oxford, UK) following the manufacturers instructions.  Briefly, 8 l of DNA at ~50 ng/l was first polished for ligation using NEBNext FFPE DNA Repair Mix.  The end-prepped DNA was then indexed by incubation with ONT Native Barcodes and Blunt/TA Ligase Master Mix (NEB).  The indexed libraries were then pooled and ligated to ONT AMXII sequencing adaptors using NEBNext Quick T4 DNA ligase.  Free adapters and short library fragments were eliminated by cleaning with AMPure beads and ONT Long Fragment Buffer.  Finally, the libraries were sequenced using a MinION_Flow Cell (R9.4.1) and a MinION-MK1C sequencer (Oxford Nanopore Technologies, Oxford, UK).