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many regions appear to be conserved, but in fact deeper sequencing would show that many of 284 those regions are not appropriate for primer design. It is advantageous therefore to include both 285 full-length as well as partial and incomplete genomes in these inclusivity datasets. However, as 286 most assay design methods attempt to maximize the number of inclusivity sequences detected 287 with the smallest number of assays, including unmodified partial sequences will force assays to 288 cover the regions that have been sequenced most often, rather than focusing on the regions of the 289 genome that are actually most conserved. While this can be a good strategy when dealing with 290 highly variable genomes for which strain diversity is better represented by available amplicon 291 sequences than available whole genome sequences, it is a poor strategy if the available amplicon 292 sequences are generated from a hypervariable region or a region that is perfectly conserved in 293 near neighbors. An alternate strategy is to “fill in” and “extend” missing sequence data by 294 interpolating and extrapolating partial and incomplete sequences (10). 295 Bacteria also present challenges for many design algorithms since they usually have circular 296 genomes without a defined starting point, and they code for proteins on both strands. As a result, 297 different sequencing labs can publish the genomes with different strands and/or starting points. 298 Thus, it is useful to perform work up front to include the same strand in the inclusivity database 299 for all members of the set. Bacteria also present challenges due to their genomic DNA size that is 300 roughly 100 times to 1000 times larger than that of viruses, thereby placing demands on 301 computational CPU (Central Processing Unit) and memory resources for signature analysis 302 algorithms (below, we describe efficient k-mer algorithms that are capable of handling bacterial 303 genomes). For bacterial inclusivity databases, it is recommended that partial genomes be 304 segregated into a separate database from the full-length genomes. Partial genomes can then be 305 avoided for purposes of design but later included in testing for coverage with an algorithm such 306

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