title: DNA-Seq of Staphylococcus aureus
design: For all specimens, detection of S. aureus was performed using routine clinical la-boratory methods. To further confirm the presence of prototypical S. aureus and to fa-cilitate detection of SCVs, a portion of each sample was also treated as follow. For spu-tum samples, 100 Lof dithiothreitol (100 mg/mL) were added to 100 mg of sputum [58,59]. From this solution, 100 L was then added to 10 mL of NaCl broth that con-sisted of 7.5 g of tryptone, 1.25 g of neutralized liver digest, 2.5 g of yeast extract, 37.5 g of NaCl, 5 g of (D-) mannitol, 2 g of glucose, and 0.5 g of thymidine in a final volume of 500 mL. For deep throat specimens, swabs were directly used to inoculate 10 mL of NaCl broth. Cultures were aerobically incubated at 35oC with shaking (225 rpm) for 48 h. Aliquots were then spread on two different media. Mannitol-salt agar (MSA) was used for detection of prototypical S. aureus while brain-heart infusion agar (BHIA) supplemented with 10 g/mL of nalidixic acid, 10 g/mL of polymyxin B and 2 g/mL of gentamicin (BHIA-SCV) was used to suppress Pseudomonas sp. and to support the growth of SCVs [58,6062].From each sample, a maximum of 8 prototypical S. aureus and SCV colonies were isolated from MSA or BHIA-SCV. Very small and non-pigmented colonies were con-sidered to be SCVs. All prototypical colonies and SCVs were confirmed to be S. aureus by PCR detection of the femA gene [63] and of the 668 pb product amplified from the nuc gene by using the following primer sequences:  nuc FWD 5-GGCATCTAGAGCTAAGTCGTGGCATATGTATGGC-3 and nuc REV 5-CCGCACTAGTCCTTGACCTGAATCAGCGTTG-3. The mecA gene coding for the low-affinity penicillin-binding protein responsible for methicillin resistance in S. aureus [64] was also identified by PCR. All isolates were kept frozen at -80oC.