Figure 1.
Estimated timelines of WGS and finishing in relation to anthrax disease progression in anthrax cases.
The anthrax disease progression and the results of antibiotic treatment in the first ten cases of the 2001 anthrax letter attack [13] and two cases of anthrax-like disease caused by B. cereus in 1996 [14]. Colored circles indicate when individual cases of inhalational anthrax from 2001 attack were started on antibiotic therapy and colored triangles indicate when 1996 B. cereus fatal pneumonia cases resembling anthrax were started on antibiotic therapy. The crosses indicate the time of death of 6 of the 12 patients. The bars in the top part of the top panel indicate the estimated timelines involved in obtaining WGS sequencing from the time the patient seeks medical care. Currently, there is no standard protocol or fixed time frame for genome finishing and the process varies from a few months to years depending on the complexity of the genome and approaches used.
Table 1.
Statistics of genome sequence of wild type and mutant B. anthracis strains.
Figure 2.
Alignment of gerH region between 34F2 and gerH::ery mutant.
Screenshot of the gerH region of B. anthracis strains 34F2 and gerH::ery mutant by Artemis comparison tool (ACT)-based genome comparison. Blue bars indicate regions sharing significant matches using the BLAST alignment tool. The genome viewer tool shows the insertion of the erythromycin gene in the gerHA gene and a small deletion within gerHA gene downstream of the insertion.
Table 2.
Location of true positive variants in the genomes of mutant B. anthracis compared to the reference genome.
Table 3.
Determination of putative variants in the genomes sequenced in this study.
Figure 3.
Sanger-verified true positive variations as a function of percent concordance and coverage.
Validation of high quality variations in the genome sequences produced by GS Reference Mapper software. The graph shows the percentage of concordance among the reads covering a variant position as a function of depth of coverage. The graph indicates all the high-quality unique variations in the eight genomic sequences obtained in this work. Circles represent false positive variations while plus signs indicate true positive variations as determined by Sanger sequencing. Squares represent identity-by-descent (IBD) variations that were not verified by Sanger sequencing as they were expected to be present in the descendant strains. The various colors of the symbols represent different strains.
Figure 4.
Proposed pipeline for rapid identification of genetic modifications in B. anthracis.
The three stages involved in genetic characterization of B. anthracis in a rapid response scenario. The first day is needed for isolation of the organism from the patient and extraction of genomic DNA. During the second day WGS will be performed using Roche 454 technology. The third day is needed to analyze the genome sequence data and figure out genetic modifications. The various bioinformatics tools used in this work are shown.