SPADA Docs

design constraints, thereby compromising design quality by introducing bias into the signature 264 regions (e.g., due to the number of times partial sequences are present, rather than focusing on 265 regions that are actually most conserved). Use of poor-quality sequences that contain deletions or 266 inserted sequences can result in assays that detect “phantom” sequences that do not exist in 267 nature. 268 The ideal case occurs when the inclusivity database fully represents the diversity of extant 269 natural (or engineered) viral pathogens with high-quality full-length genomes (e.g., Ebola, HIV, 270 and Influenza A viruses). The availability of low-cost sequencing methods has made such high- 271 quality genomes more common, though often such a ready-made, up-to-date collection does not 272 exist. Then, it is incumbent on the assay developer to gather all available sequences into a 273 curated inclusivity database taking the sequence quality into consideration (see above). Some 274 viruses have highly variable genomes (e.g., the human rhino viruses (HRV types A and B), 275 human papilloma viruses (HPV), LCMV, Lassa virus and CCHFV). For such highly variable 276 viruses, utilizing full-length genomes (and removing partial sequences) is of paramount 277 importance for high quality PCR design. 278 Alternatively, there are some viruses (e.g., Marburg virus subtypes Ci67, Musoke, and 279 RAVN) where only a few examples have been fully sequenced to date. Such cases occur with 280 newly emerging infectious diseases or diseases that have sparked little research interest. For 281 these cases, utilizing only the few full-length genomes would result in “over-fitting” wherein 282 many regions appear to be conserved, but in fact deeper sequencing would show that many of 283 those regions are not appropriate for primer design. It is advantageous therefore to include both 284 full-length as well as partial and incomplete genomes in these inclusivity datasets. However, as 285 most assay design methods attempt to maximize the number of inclusivity sequences detected 286

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