Seqsphere software version 2

Staphylococcal DNA was prepared for WGS using lysostaphin or achromopeptidase, the Maxwell^® 16 Tissue DNA Purification kit (Promega Corporation, Madison, WI) and the Genomic DNA Clean & Concentrator^™ kit (Zymo Research Corporation, Irvine, CA), performed according to the manufacturers’ instructions. WGS was performed on a MiSeq (Illumina, Inc., San Diego, CA) using the paired-end mode (2x250) in the Mayo Clinic Medical Genome Facility. Reads were trimmed using Trimmomatic. FASTQ files were imported into SeqSphere+ software version 2.3 (Ridom GmbH) for analysis. de novo assembly and application of a S. aureus cgMLST scheme was done using SeqSphere+ with default parameters and with 1,861 queried target genes, as described previously [19 (link)]. If genomes did not contain ≥95% of the 1,861 cgMLST target genes with a minimum of ten times global local coverage, WGS was repeated.

Genomic DNA was extracted and whole-genome sequencing was then performed using Illumina HiSeq 2000 sequencer or by Illumina MiSeq sequencer. After sequencing, the reads were quality-trimmed and then assembled using the CLC Genomics Workbench software version 6.0 (CLC bio, Aarhus, Denmark). The resulting assembly files were exported as ACE files and imported into SeqSphere⁺ software version 2.3 (Ridom GmbH, Münster, Germany). The assembly contigs together with epidemiologic meta-data were also deposited to the Neisseria BIGSdb website [22 (link)].
A core genome multi-locus sequence typing (cgMLST) target set was determined using all finished N. meningitidis genomes available in GenBank as of February 2014 (n = 14) [23 (link)]. The genome of strain FAM18 (NC_008767) was used as a reference. Complete sequence for each gene was analyzed in comparison to the FAM18 reference. The combination of all alleles in each strain formed an allelic profile. The exported aligned sequences were then imported into MEGA6 and a neighbor-joining tree was generated with default parameters and the bootstrap option (with 1.000 replications) turned on [24 (link)]. All classical typing results achieved by Sanger sequencing were confirmed by WGS data analysis [25 (link)].