N315 ORFs BLASTn
We performed a whole-genome comparison based on the nucleotide identity between ORFs from a reference genome and genomes from CC5 to identify differences between the RdJ clone and related lineages.
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We used a gene-based genomic approach to assess the genomic changes that occurred throughout the evolution of ST105 (RdJ). For this analysis, all annotated coding sequences from the genome of the Brazilian strain MRSA n315 were obtained using Geneious Prime software (Tool: extract annotations) and concatenated in one single fasta file. The concatenated file was used as a query against all genomes in a database composed of 661 CC5. Genome identifications were plotted in the Excel file, with the associated lineage identification (MLST). Genomes were organized to comprise all ST105-RdJ together in the latter section of the list, and non-ST105(non-RdJ) genomes were in the basal section. This database was created with genomes sequenced by our group and others downloaded from Genbank NCBI. Genomes were uploaded to Geneious Prime and exported in a single fasta file. The database was created by command line using BLASTn command line application (https://www.ncbi.nlm.nih.gov/books/NBK279690/). (command: makeblastdb - in nameofthefile.fasta -dbtype nucl). The ORFs from N315 were used as query, by the command: blastn -query ORFSN315.fasta -db nameofthefile.fasta -outfmt "6 qseqid sseqid pident qcovs" > results.xls Hits with less than 90% of query coverage were excluded. Factor “1” was designed for the category comprehending BLASTn hits with 100% identity and the factor “0” for that including any hit between 0 and <100% identity. Fold-change and T-test were calculated to compare the distribution of the binary factors for each ORF in the group of genomes representing RdJ clone and all other genomes from the list. A volcano graphic was plotted using the negative log10 of the p-value and the log10 of the fold-change. A cutoff of 50 for the -log10(p-value) was set and specific ORFs were selected for further analysis (graphic not shown here).