SPADA Draft Documents

labels, clinical severity and geographic origin) or incomplete metadata that can lead to a 244 sequence being incorrectly included in, or excluded from, the inclusivity data. 245 It is recommended to include only high-quality sequences in the inclusivity database, since 246 including poorly determined sequences can effectively reduce the number of conserved 247 signatures regions in a set of target genomes. Use of partial sequences in the inclusivity can 248 cause assay design algorithms to ignore otherwise promising regions. Use of poor-quality 249 sequences that contain deletions or inserted sequences can result in assays that detect “phantom” 250 sequences that do not exist in nature. 251 The ideal case occurs when the inclusivity database fully represents the diversity of extant 252 natural (or engineered) pathogens with high-quality full-length genomes (e.g., Ebola, HIV, and 253 Influenza A viruses). The availability of low-cost sequencing methods has made such high- 254 quality genomes more common, though often such a ready-made up-to-date collection is not 255 available, and it is incumbent on the assay developer to gather all available sequences into a 256 curated inclusivity database. In such a case, it is preferred to not include partial sequences in the 257 inclusivity set because partial sequences introduce artificial design constraints, thereby 258 compromising the design quality by introducing bias into the signature regions (e.g., due to the 259 number of times partial sequences are present rather than focusing on regions that are actually 260 most conserved). Some viruses have highly variable genomes (e.g., the human rhino viruses 261 (HRV types A and B), human papilloma viruses (HPV), LCMV, Lassa virus and CCHFV). For 262 such highly variable viruses utilizing full-length genomes (and removing partial sequences) is of 263 paramount importance for high quality PCR design. 264 Alternatively, there are some viruses (e.g., Marburg virus subtypes Ci67, Musoke, and 265 RAVN) where only a few examples have been fully sequenced to date. Such cases occur with 266

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