VNTR markers.

<p>*Data obtained in this study.</p>†<p>The individual marker diversity (D) was calculated as D = [1-∑(allele frequency)²].</p>††<p>Location within an open reading frame.</p><p>VNTR markers.</p

<i>PmeI</i> pulsed-field gel electrophoresis (PFGE) patterns for <i>F. tularensis</i> subsp. <i>holarectica</i>.

<p>Polymorphic band position 1 consists of two fragments in PFGE type 2. Polymorphic band position 2 and 3 consist of two fragments at the same position, both missing in PFGE type 2. Polymorphic band position 6 consists of two fragments in PFGE type 3.</p

Agarose-MAMA primers targeting 18 previously published SNP positions.

<p>Agarose-MAMA primers targeting 18 previously published SNP positions.</p

Size differences in allele-specific PCR products.

<p>A) Schematic of PCR amplicons originating from ancestral and derived genomic templates. For both PCR amplicons, gray represents sequence originating from the primers (forward and reverse) and synthesized internal sequence from PCR extension. The primer sequence and synthesized internal sequence within the amplicon are indicated by the bracket. For the derived amplicon, the blue represents the incorporated GC-clamp which originates from the 5’end of the derived MAMA forward primer. The SNP region for each amplicon is represented in green (ancestral) and yellow (derived) and the deliberate antepenultimate mutation is represented as red (ancestral) and light blue (derived). B) Allele-specific PCR products migrate at different rates on a 2% agarose gel due to their size difference as conferred by the GC-clamp.</p

Flow chart providing the hierarchical organization of 18 SNP-genotyping assays.

<p>The hierarchy provides the sequential order to allow for stepwise identification of the genotype of an unknown isolate. Genetic subgroups are represented by colored circles labeled with each genetic subgroup. Individual assays are represented by the black bar labeled with each assay ID. The derived and ancestral allele states for each assay are indicated immediately above and below the black bar.</p

The SNP allele state profiles of genetic subgroups targeted for agarose MAMA design where the Derived SNP state is shaded and Ancestral state is unshaded.

<p>The SNP allele state profiles of genetic subgroups targeted for agarose MAMA design where the Derived SNP state is shaded and Ancestral state is unshaded.</p

Mad-05 MAMA tool showed congruent genotyping results when generated across two research institutes using identical DNA samples.

<p>Side by side comparison of Agarose-MAMA gel images generated at NAU (top) and IPM (bottom) using different reagents and instruments. <i>Y</i>. <i>pestis</i> sample names follow the designation scheme practiced at IPM. Individual samples are designated an ID # according to consecutive order of collection per a given year. Strain 87/11 was the 87^th isolate collected in 2011.</p

Agarose-MAMA is capable of genotyping <i>Y</i>. <i>pestis</i> directly from complex clinical samples if pathogen targets are at sufficient levels.

<p>(A) Agarose-MAMA (Mad-43) gel showing the PCR products for two template controls at the expected size for each respective ancestral and derived allele state (#1 and #2, respectively). Three complex clinical samples (#3-#5) also yielded PCR products with the size expected of an ancestral genotype. Two other complex clinical samples (#6 & #7) showed no PCR products but displayed a banding pattern consistent with NTC negative controls. (B) To assess the relative quantity of the <i>Y</i>. <i>pestis</i> target in the five clinical samples (#3, #4, #5, #6, #7), we generated amplification plots of these clinical samples on a TaqMan 3a assay used to target <i>Y</i>. <i>pestis</i> chromosomal DNA. Three clinical samples (#3-#5) showed amplified at a mid-range cycle-time (Ct) value consistent with high copy numbers of template DNA [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006077#pntd.0006077.ref021" target="_blank">21</a>]. These same samples showed a robust signal of a PCR product on the MAMA gel. The two samples (#6 & #7) that failed on the MAMA gel showed a high Ct value and failed amplification, respectively, when tested by real-time PCR. The pairing of TaqMan 3a assay with MAMA gel results on the same templates provided insight to the genotyping capability of MAMA tools on complex clinical samples with low-level target template.</p

Simplified SNP phylogeny of <i>Y</i>. <i>pestis</i> depicting subgroups identified in Madagascar.

<p>Colored circles indicate phylogenetic groups as previously described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006077#pntd.0006077.ref035" target="_blank">35</a>][<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006077#pntd.0006077.ref011" target="_blank">11</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006077#pntd.0006077.ref034" target="_blank">34</a>]. Group names are assigned as letters and sometimes followed by a number (e.g. s4). Black bars indicate the phylogenetic positions of the 18 SNPs targeted for MAMA PCR design in this study.</p