Whole Genome Sequencing of Streptococcus pneumoniae

Genomic DNA preparation, library construction, whole genome sequencing (WGS), and bioinformatics pipeline features have been previously described (Li et al., 2016 (link), 2017 (link); Metcalf et al., 2016a (link),b (link)). Streptococcus pneumoniae strains were cultured on Trypticase soy agar supplemented with 5% sheep blood and incubated overnight at 37°C in 5% CO₂. Genomic DNA for short-read WGS was extracted manually using a modified QIAamp DNA mini kit protocol (Qiagen, Inc., Valencia, CA, USA). Nucleic acid concentration was quantified by an Invitrogen Qubit assay (Thermo Fisher Scientific Inc., Waltham, MA, USA) and samples were sheared using a Covaris M220 ultrasonicator (Covaris, Inc., Woburn, MA, USA) programmed to generate 500-bp fragments. Libraries were constructed on the SciCloneG3 (PerkinElmer Inc., Waltham, MA, USA) using a TruSeq DNA PCR-Free HT library preparation kit with 96 dual indices (Illumina Inc., San Diego, CA, USA) and quantified by a KAPA qPCR library quantification method (Kapa Biosystems Inc., Wilmington, MA, USA). WGS was generated employing two MiSeq instruments and the MiSeq v2 500 cycle kit (Illumina Inc).