Computational protocol: Induction of Type I Interferon Signaling Determines the Relative Pathogenicity of Staphylococcus aureus Strains

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Protocol publication

[…] Genomic DNA was prepared from overnight cultures of 502A strain (RN6607 BK21236 NRS149) using the Tissue DNeasy kit (Qiagen) as above. Library preparation and sequencing was done using the Ion Torrent Personal Genome Machine 316 chip, performed by the Columbia Genome Center using manufacturer-specified protocols. Sequence assembly was performed with Newbler (http://454.com/products/analysis-software/index.asp), generating 181 total contigs (N50 = 25, median contig = 2955). The total size of the contigs was 2,740,204 bp. Preliminary Annotation was done using the Rapid Annotation using Subsystem Technology (RAST) server (http://rast.nmpdr.org). We focused our initial analysis on known S. aureus virulence factors. To create a heat map representation of selected genes we used BLAST-blast analysis (http://blast.ncbi.nlm.nih.gov) using default settings on the megablast algorithm and surveying the nr/nt databases. Query sequences were from the USA300 TCH1516 genome sequence or the 502A sequence as noted.Selected S. aureus genomes were used to create a whole genome phylogenetic matrix of gene coding regions using the Ortholog ID process . The matrix was further pruned to include only genes found in more than 96% of known S. aureus genomes, and includes only variable nucleotide positions. The final matrix had 93,249 characters. Phylogenies were constructed using PAUP 4b10 (http://paup.csit.fsu.edu) using the maximum parsimony criterion with random addition (RA) followed by tree bisection and reconnection (TBR), Nreps = 1000, with characters and character state transformations given equal weight. Node support was calculated based on 1000 bootstrap pseudoreplicates using the heuristic search strategy above. […]

Pipeline specifications

Software tools Newbler, RAST, BLASTN, PAUP*
Application Phylogenetics
Organisms Mus musculus, Staphylococcus aureus, Epipremnum aureum, Homo sapiens
Diseases Infection, Pneumonia, Pneumonia, Staphylococcal, Soft Tissue Injuries