WGS of Mycobacterium Ulcerans: 1173.15
The isolate was cultivated on Lowenstein-Jensen medium for 5 months. Prior to DNA extraction, it was grown on Middlebrook 7H10 agar and enriched with an OADC (oleic acid, albumin, dextrose, and catalase) supplement. Cells were then scraped from the agar and washed with wash solution (0.3 M sucrose, 50 mM Tris pH 8.0, and 10 mM EDTA). Every 50 mg of cells were treated with 3.5 mM lysozyme and 0.4 mM RNase A in 20-L volume overnight at 37C, followed by centrifugation. The pellets were resuspended in 1  Tris/EDTA (TE) buffer, containing 36 nM proteinase K and 1% SDS. After incubation for 1 h at 50C and another centrifugation, the pellets were resuspended in lysis solution (6% guanidine hydrochloride, 1% Tween 20, and 1% Nonidet P-40) and incubated for 1 h at 37C. Finally, the cells were completely lysed with a bead beater, and the aqueous phase of the lysates was extracted with chloroform. DNA was precipitated with isopropanol/3 M sodium acetate on ice. The precipitates were washed with 70% ethanol, dried, and dissolved in 1  TE buffer. DNA was fragmented (200 to 300 bp) using a Bioruptor Standard (Diagenode) device. Sequencing libraries were prepared from 1 g of fragments using a NEBNext Ultra DNA library prep kit for Illumina (NEB) and sequenced on a MiSeq sequencer (Illumina)