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191 Figure 4. Traditional vs. modern assay development pipeline. Inc/Exc: Inclusivity/Exclusivity.

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4.0 Assay Design

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4.1 Target selection: user defined vs unbiased (in silico)

Traditionally, assay target selection has been an ill-defined process that is strongly influenced 196 by the preferences and experience of individual assay designers. Often, assay targets are selected 197 from lab-specific research interests on specific genes of given pathogens or from known 198 (literature-based), or suspected, virulence factor genes. The resulting assays are then screened 199 (either computationally or “by eye”) for inclusivity and exclusivity using hand-selected 200 sequences. The resulting assays are also often broadly screened computationally against all 201 known sequences (e.g., by using Basic Local Alignment Search Tool ( BLAST ) to identify 202

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