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

BioLaboro architecture 158 BioLaboro is comprised of three algorithms -BioVelocity, Primer3, and PSET -which 159 are built into a pipeline for user-friendly applications. The user has the option to launch one of 160 four different job types: Signature Discovery, Score Assay Targets, Validate Assay, or New 161 Assay Discovery. Each of the three algorithms can be run individually or together as a complete 162 end-to-end pipeline (Figure 1 ). For the BOMV use case, in the first phase of the pipeline 163 BioVelocity was used to analyze a set of genome sequences for unique regions that are both 164 conserved and signature to the target sequences selected. This was achieved by splitting a chosen 165 representative whole genome sequence into sliding 50 base pairs (bps) k-mers. Each k-mer was 166 then scanned against all target sequences to determine conservation. Conserved k-mers were then 167 elongated based on overlaps and formed into contigs. These contigs were then split into k-mers ≤ 168 250 bps and scanned against all non-target sequences to determine specificity. All passing 169 sequences were then elongated based on overlaps and the signature contigs were passed to the 170 next step in the pipeline. Primer3 was then used to evaluate the signature contigs to identify 171 suitable primers and probes for assay development. Primer3 was run in parallel against all 172 signatures and the output was ranked by penalty score in ascending order. The top five best 173 primer sets were passed along to the final step in the pipeline, PSET. In this step the primer sets 174 were run through a bioinformatics pipeline which aligned the sequences against large public 175 sequence databases from NCBI using BLAST and GLSEARCH [36] to determine how well each 176 assay correctly aligned to all target sequences while excluding off-target hits. 199 Even the two assays that passed in silico criteria did not have perfect matches raising the 200 possibility that these assays may fail in wet lab testing due to mismatches against currently 201 available BOMV genomic sequences. Hence, as described below, we designed new assays using 202 the BioLaboro platform.