Whole Genome Analysis of Diverse Sequence Types Advances Our Understanding of Population Genetics and Evolution of Staphylococcus aureus
DNA samples were prepared for multiplexed, paired-end sequencing following the manufacturers protocol. For each isolate, 1-5µg dsDNA in 200µl was sheared and then purified using the QIAquick PCR Purification kit (Qiagen). Enzymatic processing of the DNA followed the guidelines as described in the Illumina protocol, with the exceptions of processing enzymes obtained from New England Biolabs (New England Biolabs, Ipswich, MA) and oligonucleotides and adaptors from Illumina (Illumina Inc). After ligation of the adaptors, the DNA was run on a 2% agarose gel for 2 hours; subsequently a gel slice containing 500-600 bp fragments of each DNA sample was isolated and purified using the QIAquick Gel Extraction kit (Qiagen). Individual libraries were quantified with qPCR on the ABI 7900HT (Life Technologies Corp.) using the Kapa Library Quantification Kit (Kapa Biosystems, Woburn, MA). Based on the individual library concentrations, equimolar pools of twelve S. aureus libraries were prepared at a concentration of at least 1nM. To ensure accurate loading onto the flowcell, the same quantification method was used to quantify the final pools. The pooled libraries were sequenced on the Illumina GAIIx using “Genomic DNA sequencing primer V2” for 36 cycles.