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conservation of the assay signatures to the target strains in the inclusivity panel and their low 127 conservation in the exclusivity panel, the “brute force” testing of all available strains is not cost 128 effective. As described below, a cost-effective selection of inclusivity and exclusivity strains for 129 testing can be guided by in silico analyses and in silico PCR testing (section 2.2). 130

131 Figure 2. Signature sequence identities of the target sequences for inclusivity/exclusivity panel 132 strains (SPADA panels). Perfect (x) refers to a hypothetical assay; BA: Bacillus anthracis , YP: 133 Yersinia pestis , FT: Francisella tularensis ; BK-P: Burkholderia pseudomallei , BK-M: 134 Burkholderia mallei , BK-NN: Burkholderia near neighbors. Representative data set depicting the 135 heat map of amplicon percentage identity in various whole genome sequences of bacterial strains 136 used in inclusivity and exclusivity testing of molecular assays. Strains are numbered as columns 137 from 1 up to 24. Each row (indicated by lower case letters) represents a given assay.

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