BioLaboro: A bioinformatics system for detecting molecular assay signature erosion and designing new assays in response to emerging and reemerging pathogens

Mitchell Holland; Daniel Negrón; Shane Mitchell; Nate Dellinger; Mychal Ivancich; Tyler Barrus; Sterling Thomas; Katharine W. Jennings; Bruce Goodwin; Shanmuga Sozhamannan

In the third phase, PSET was used to test the five newly designed assays identified by 250 Primer3 in silico against publicly available sequences. PSET was used to analyze the primers and 251 probes in the new assays using bioinformatics tools to identify potential false-positive and false-252 negative matches to NCBIs BLAST nucleotide sequence repositories (nt, gss, and env_nt) 253 comprised of over 220 million sequences (as of last update in September 2019). BLAST+ was 254 used to compare the assay amplicon sequences against these sequence repositories to identify 255 matches. These matches were then used to create a custom library of sequences for GLSEARCH, 256 a global-local sequence comparison tool in the FASTA suite of programs, which was used to 257 search for the individual primers and probes. The resulting output was then processed and 258 filtered based on pre-defined hit acceptance criteria. These criteria require that the assay 259 components all hit to 90% identity over 90% of the component length, primer pairs were on 260 opposite strands, and the total amplicon size was no greater than 1000 bps. The results were then 261 validated by comparing the hits to the target NCBI Taxonomy identifier (ID), and true and false 262 matches were reported. PSET results confirmed that the top five primer sets had true positive hits 263 to all three BOMV genomes and no false positive hits to any other organism (Table 4) .