Library preparation kit with standard pcr library amplification

Blood samples from patients were plated and single colonies picked and stored at −80 °C until processing. Samples were grown on trypticase soy agar (TSA) (BD, Franklin Lakes, USA) at 37 °C for 24 h, after which DNA was extracted using the Qiagen DNeasy Blood and Tissue Purification kit including a pre-lysis step using lysostaphin for Gram-positive extractions according to the manufacturer’s recommendations (Qiagen, Valencia, CA, USA). DNA was prepared for multiplexed, paired-end sequencing (2×100 bp) on an Illumina GA_IIX instrument using the Library Preparation kit with standard PCR library amplification (KAPA Biosystems, Woburn, MA, USA) as described previously [22 (link)]. Additionally, six samples that failed initial sequencing were resequenced on an Illumina MiSeq instrument using 2×250 V2 technology (Table S1). The average coverage across all 100 isolates was 130×. Genomes were assembled with UGAP (https://github.com/jasonsahl/UGAP), which utilizes SPAdes v3.10.1 [23 (link)] and Pilon [24 (link)] post-assembly error correction. MLST was performed with the raw reads using SRST2 [25 (link)].

Isolates were grown on trypticase soy agar media and DNA was prepared using DNeasy Blood & Tissue Kit as described by the manufacturer (Qiagen, Valencia, CA) with the addition of lysostaphin to the gram-positive extraction protocol from Qiagen. The DNA samples were prepared for multiplexed, paired-end sequencing with a 500 base pair insert using Library Preparation Kit with Standard PCR Library Amplification (KAPA Biosystems, Woburn, MA). A 100 bp read paired-end run was used for all twenty-four isolates on the GAIIx sequencing platform (Illumina, Inc. San Diego, CA).