Single colonies from each isolate regrown on LB plates were inoculated into LB medium and grown overnight at 37°C with 250 rpm shaking. Cell pellets were frozen at −20°C following centrifugation of 0.5 ml of culture at 16 000 x g for 2 min and removal of the supernatant. Genomic DNA was extracted from cell pellets using the PureLink Genomic DNA Mini Kit (Life Technologies) following the manufacturer's instructions, and was further concentrated and purified by ethanol precipitation as previously described (24 (link)). Genomic DNA concentrations used to calculate input DNA for library preparation (200 ng in 55 μl volume) were quantified using a Qubit broad-range dsDNA assay (Invitrogen). Libraries were prepared for sequencing using the TruSeq Nano HT Library Preparation Kit (Illumina).
Sequence variants were determined from sequencing fastq output files using breseq (25 (link)) version 0.24rc6 and NCBI Reference Sequence NC_000913.3 as the reference sequence, with a base quality cutoff of 20. All called mutations were present in >80% of the spanning reads and in regions with coverage of more than 20-fold, with the exception of a nagA A203E mutation which did not meet these criteria but was detected in strain mutS_pMA1_NaCl_20.
Sequence variants were determined from sequencing fastq output files using breseq (25 (link)) version 0.24rc6 and NCBI Reference Sequence NC_000913.3 as the reference sequence, with a base quality cutoff of 20. All called mutations were present in >80% of the spanning reads and in regions with coverage of more than 20-fold, with the exception of a nagA A203E mutation which did not meet these criteria but was detected in strain mutS_pMA1_NaCl_20.