Whole genome sequencing of Chlamydia trachomatis mutants resistant to a novel antichlamydial compound
Whole genome sequencing of Chlamydia trachomatis mutants resistant to a novel antichlamydial compound
The bacteria were isolated from Vero cell cultures infected with the respective strains. In brief, the infected cells were lysed by incubation in H2O, followed by sonication. Remaining intact cells and cell nuclei were removed by centrifugation at low speed (1600 rpm, 10 min). Released bacteria were then collected by centrifugation at high speed (17,000 x g, 15 min), resuspended in SPG (sucrose-phosphate-glutamate) buffer (75 g/l sucrose, 0.5 g/l KH2PO4, 1.2 g/l Na2HPO4, 0.72 g/l glutamic acid, pH 7.5), and treated for 1 hour at 37°C with DNase I (NEB) to remove host DNA. Bacteria were then washed once with phosphate buffered saline (PBS), resuspended in a small volume of PBS, followed by heat-inactivation of residual DNase I at 75°C for 20 min. Bacterial genomic DNA was extracted from the isolated bacteria using the DNeasy Blood and Tissue kit (Qiagen) and quantified using the Qubit dsDNA BR Assay kit (Thermo Fisher Scientific). A total amount of 0.2 µg DNA per sample was used as input material for the DNA library preparations. Briefly, the genomic DNA sample was fragmented by sonication to a size of 350 bp. Then DNA fragments were end-polished, A-tailed, and ligated with the full-length adapter for Illumina sequencing, followed by further size selection and PCR amplification. PCR products were purified by AMPure XP system (Beverly, USA). Subsequently, library quality was assessed on the Agilent 5400 system (Agilent, USA) and quantified by qPCR.