Analysis of the MRSA252 Genome Figure 3. S. aureus MRSA252 and Listeria monocytogenes chromosomes share near identical copper ATPase transporter and copper oxidase genes. A) Shown is a Listeria monocytogenes DNA (4,680 bp) – MRSA252 pairwise enhanced BLAT (eBLAT) DNA alignment revealing near sequence identity that spans the ORFs for the copper ATPase transporter (SAR0720; upper box) and the copper oxidase genes (SAR0721; lower box) [23]. The Listeria genomic region of this eBLAT (GenBank accession AARR00000000), used as the reference sequence, was selected as ‘other species’ in the sequence input page of EvoPrinter. Use of this feature allows for the comparative analysis of any sequence with respect to the genomes in the EvoPrinter database. Red-colored letters indicate unique bases that not shared with MRSA252. Green-colored sequences are common to both Listeria and MRSA252. B) Listeria DNA (same sequence as in panel A) eBLAT alignment with the S. aureus USA300 genome identifies CopA (SAUSA300_0078) but with a significantly lower sequence homology when compared to the MRSA252 DNA (A). doi:10.1371/journal.pone.0003074.g003 ninds.nih.gov/). Unlike other multi-genome comparative tools that display columns of aligning bases with gaps to optimize alignments, EvoPrinter displays, in a single uninterrupted view, DNA sequences that are either conserved, unique or uniquely shared, including single nucleotide polymorphisms (SNPs), as they exist in the genome or MGE of interest. Because EvoPrinter readouts show only the input reference DNA sequence and not the aligning regions of the multiple genomes included in the analysis, more sequence can be displayed in a single view than is possible with conventional multi-genome alignments. The following algorithms were developed to help identify putative HGT DNA sequences: (1) an EvoUnique profile highlights unique or uniquely shared sequences among subsets of genomes that are otherwise absent from the other genomes included in the analysis; (2) a repeat finder detects putative MGE sequences based on the repetitive presence of their sequences within bacterial chromosomes; (3) an EvoDifferences profile portrays, in a single view, those sequences that are detected in all but one of the genomes included in the analysis, and (4) input reference DNA exchange allows for re-initiation of the comparative analysis using the aligning region of another genome, thus facilitating the search for unique differences among the genomes included in the analysis. EvoPrinterHD also includes algorithms that identify sequence rearrangements in the aligning regions of the test genomes. For Staphylococcus chromosome analysis, after inserting the DNA sequence to be analyzed (the reference sequence) into the DNA sequence input window, the alignment algorithms automatically generate 9 eBLAT alignments for each of the 17 staphylococcal genomes and then assembles composite eBLAT (ceBLAT) alignments for the top three homology scoring regions of each genome. The alignment process takes seconds to complete and allows the user to examine the input DNA for repetitive sequences and to view the alignment results, in the form of an alignment scorecard [22]. The alignment scorecard gives information regarding the extent of homology to each of the test species for the top scoring alignment, and the second and third most significant alignments. Accessible from the alignment scorecard are the eBLAT alignments to the top three aligning regions for each pairwise analysis, and a ceBLAT that superimposes the top three alignments. From the scorecard, the user can select or deselect different alignments to be used in the EvoPrint analysis. The complete EvoUnique prints of the S. aureus MRSA252 and S. aureus RF122 genomes are also available online through links provided in the EvoPrinterHD bacterial genome resources section. Genomic DNA sequence files The 17 Staphylococcus genomes were curated from databases listed below. The following Staphylococcus genome sequence files were curated from the BacMap database of University of Alberta (http://wishart.biology.ualberta.ca/BacMap/): S. aureus COL, S. aureus JH1, S. aureus JH9, S. aureus MRSA252, S. aureus MSSA476, S. aureus Mu3, S. aureus Mu50, S. aureus MW2, S. aureus N315, S. aureus NCTC8325, S. aureus Newman, S. aureus RF122, S. aureus USA300, S. epidermidis ATCC12228, S. epidermidis RP62A and S. haemolyticus JCSC1435. The genome sequence files for S. aureus subsp. aureus JH1, S. aureus subsp. aureus JH9, S. aureus Mu3, and S. aureus subsp. aureus str. Newman were curated from the European Bioinformatics Institute of the European Molecular Biology Laboratory (http://www.ebi.ac.uk/genomes/ bacteria.html). The pTZ2162 plasmid sequence was obtained from the NCBI GenBank nucleotide sequence database. Genomic indexing Each of the genomes was parsed into non-overlapping K-mers three different ways and held in memory. In addition to the original 11-mer index of BLAT [47], EvoPrinterHD indexes each PLoS ONE | www.plosone.org 6 August 2008 | Volume 3 | Issue 8 | e3074