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potential false positive organisms, but this exhaustive approach is not optimal because it detects 202 many hits that are not relevant to sequences that might be found in relevant sample matrices 203 (e.g., body fluids or soil). In other words, screening primers against all known sequences is 204 overly restrictive to proper design. In contrast, a modern approach uses an unbiased search of all 205 available sequence data for the organism of interest to identify potential targets and then 206 validates those targets/genes against well-defined inclusivity, exclusivity, and environmental 207 background sequence panels (e.g., SPADA environmental panel list of organisms). 211 relatively neglected compared to other parts of the process such as instrumentation, enzymes, 212 buffer additives, and data analysis. However, high quality primer design offers a tremendous 213 opportunity to improve diagnostic performance (i.e. sensitivity, specificity, and limit of 214 detection), as well as reduce the development time and cost. Figure 5 shows a traditional primer 215 design approach. Such a design pipeline brings together numerous tools that work well for their 216 intended uses, but were not specifically optimized to be used together for primer design. As a 217 result of the deficiencies of such traditional approaches, developing a high-performing assay 218 requires extensive experimentation with numerous cycles of re-design and testing and even after 219 significant financial investment, the resulting assays are often fragile and prone to failure (8). 220 Below, modern methods are recommended for each step in the assay development pipeline. 221 These methods are database-driven, apply physical chemistry modeling, and utilize modern 222 design algorithms and computational resources to overcome some of the weaknesses associated 223 with the traditional approach. 224 208 209 4.2 Traditional primer design paradigm 210 Primer design is a critical aspect in the development of diagnostic assays that has been

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