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22Background: Emerging and reemerging infectious diseases such as the novel Coronavirus 23 disease, COVID-19 and Ebola pose a significant threat to global society and test the public 24 health community's preparedness to rapidly respond to an outbreak with effective diagnostics 25 and therapeutics. Recent advances in next generation sequencing technologies enable rapid 26 generation of pathogen genome sequence data, within 24 hours of obtaining a sample in some 27 instances. With these data, one can quickly evaluate the effectiveness of existing diagnostics and 28 therapeutics using in silico approaches. The propensity of some viruses to rapidly accumulate 29 mutations can lead to the failure of molecular detection assays creating the need for redesigned 30 or newly designed assays. 31Results: Here we describe a bioinformatics system named BioLaboro to identify signature 32 regions in a given pathogen genome, design PCR assays targeting those regions, and then test the 33 PCR assays in silico to determine their sensitivity and specificity. We demonstrate BioLaboro 34 with two use cases: Bombali Ebolavirus (BOMV) and the novel Coronavirus 2019 (SARS-CoV-35 2). For the BOMV, we analyzed 30 currently available real-time reverse transcription-PCR 36 assays against the three available complete genome sequences of BOMV. Only two met our in 37 silico criteria for successful detection and neither had perfect matches to the primer/probe 38 sequences. We designed five new primer sets against BOMV signatures and all had true positive 39 hits to the three BOMV genomes and no false positive hits to any other sequence. Four assays 40 are closely clustered in the nucleoprotein gene and one is located in the glycoprotein gene. 41

Background The SARS-CoV-2 Delta variant was first identified in the U.S. in March 2021 and has rapidly become the predominant lineage across the U.S. due to increased transmissibility, immune evasion and vaccine breakthrough. The aim of this study was to better understand the genetic diversity and the potential impact of mutations observed in SARS-CoV-2 viruses circulating in the U.S. in vaccinated individuals. Results Whole genome sequencing was performed on thirty-four SARS-CoV-2 positive samples using the Oxford Nanopore MinION. Evolutionary genomic analysis revealed two novel mutations, ORF1b:V2354F and a premature stop codon, ORF7a:Q94*, identified in a cluster of SARS-CoV-2 Delta isolates collected from vaccinated individuals in Colorado. The ORF1b:V2354F mutation, corresponding to NSP15:V303F, may induce a conformational change and result in a disruption to a flanking beta-sheet structure. The premature stop codon, ORF7a:Q94*, truncates the transmembrane protein and cytosolic tail used to mediate protein transport. This may affect protein localization to the ER-Golgi. In addition to these novel mutations, the cluster of vaccinated isolates contain an additional mutation in the spike protein, at position 112, compared to the Delta variant defining mutations. This mutation, S112L, exists in isolates previously obtained in the U.S. The S112L mutation substitutes a bulky hydrophobic side chain for a polar side chain, which results in a non-conservative substitution within the protein that may affect antibody-binding affinity. Additionally, the vaccinated cluster of isolates contains non-synonymous mutations within ORF8 and NSPs which further distinguish this cluster from the respective ancestral Delta variant. Conclusions These results show there is an emerging sub-lineage of the ancestral Delta variant circulating in the U.S. As mutations emerge in constellations, those with a potentially beneficial advantage to the virus may continue to circulate while others will cease.

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays are the most widely used molecular tests for the detection of SARS-CoV-2 and diagnosis of COVID-19 in clinical samples. PCR assays target unique genomic RNA regions to identify SARS-CoV-2 with high sensitivity and specificity. In general, assay development incorporates the whole genome sequences available at design time to be inclusive of all target species and exclusive of near neighbors. However, rapid accumulation of mutations in viral genomes during sustained growth in the population can result in signature erosion and assay failures, creating situational blind spots during a pandemic. In this study, we analyzed the signatures of 43 PCR assays distributed across the genome against over 1.6 million SARS-CoV-2 sequences. We present evidence of significant signature erosion emerging in just two assays due to mutations, while adequate sequence identity was preserved in the other 41 assays. Failure of more than one assay against a given variant sequence was rare and mostly occurred in the two assays noted to have signature erosion. Assays tended to be designed in regions with statistically higher mutations rates. in silico analyses over time can provide insights into mutation trends and alert users to the emergence of novel variants that are present in the population at low proportions before they become dominant. Such routine assessment can also potentially highlight false negatives in test samples that may be indicative of mutations having functional consequences in the form of vaccine and therapeutic failures. This study highlights the importance of whole genome sequencing and expanded real-time monitoring of diagnostic PCR assays during a pandemic.

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