SPADA Meeting Book

2.1 Additional considerations with the status quo testing of assays against inclusivity/exclusivity 103 panels 104 The Stakeholder Panel on Agent Detection Assays (SPADA) inclusivity/exclusivity panels 105 for the biodefense relevant bacterial pathogens, such as Bacillus anthracis , Yersinia pestis , 106 Brucella suis , Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis , 107 comprise a total of approximately 100 strains. These strains are used to validate the 108 inclusivity/exclusivity criteria for the respective detection assays. Most of the inclusivity strains, 109 and some exclusivity strains, are considered Biosafety Level 3 (BSL3) agents and, as a result, are 110 limited to laboratories that are registered and certified for such work. Moreover, extensive 111 laboratory testing adds cost and time to the assay development effort. 112 Many whole genome sequences of these bacterial strains are available now (2-6), which 113 allows the in silico evaluation of assays. An example set of assays developed prior to the next 114 generation sequencing revolution with representative analyses is illustrated in Figure 2. As 115 expected, the majority of the evaluated assay signatures had perfect sequence matches to the 116 target inclusivity genome sequences, and much less (0 % to 40 %) sequence identity to the 117 exclusivity panel genome sequences. However, for all the assays evaluated, there was no 118 “perfect” assay (i.e., no false positives and no false negatives). Some assays were 119 computationally predicted to have both false negatives (e.g., Bacillus anthracis assay 1 against 120 strain 10 in the inclusivity panel) and false positives (e.g., Bacillus anthracis assay 1 against 121 strain 8 in the exclusivity panel). Many of these predicted assay failures correspond to expected 122 deviations based on the genotypes of these strains. There are other assays that simply fail the 123 inclusivity and/or exclusivity criteria (e.g., Bacillus anthracis assay 7 or Yersinia pestis assay 15) 124 and are therefore not reliable diagnostics due to low specificity. However, given the high 125

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