Host, Viral, and Environmental Transcriptome Profiles of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Daniel J Butler; Christopher Mozsary; Cem Meydan; David C Danko; Jonathan Foox; Joel Rosiene; Alon Shaiber; Ebrahim Afshinnekoo; Matthew MacKay; Fritz J Sedlazeck; Nikolay A Ivanov; Maria A Sierra; Diana Pohle; Michael Zeitz; Vijendra Ramlall; Undina Gisladottir; Craig D Westover; Krista Ryon; Benjamin Young; Chandrima Bhattacharya; Phyllis Ruggiero; Bradley W Langhorst; Nathan A Tanner; Justyn Gawrys; Dmitry Meleshko; Dong Xu; Jenny Xiang; Angelika Iftner; Daniela Bezdan; John Sipley; Lin Cong; Arryn Craney; Priya Velu; Ari Melnick; Iman A Hajirasouliha; Thomas Iftner; Mirella Salvatore; Massimo Loda; Lars F Westblade; Shawn Levy; Melissa Cushing; Nicholas P Tatonetti; Marcin Imielinski; Hanna Rennert; Christopher Mason

Primers were designed using PrimerExplorer (v4.0), as per guidelines in Zhang et al., 2020 . This specifically utilized the LAMP-compatible primers for the on the COVID-19 reference genome (NCBI). LAMP's inherent specificity (using 4-6 primers vs. 2 for PCR amplification) in combination with this in-silico analysis revealed there is limited opportunity for cross-reactivity to allow for false-positive reporting or affect performance of the N-gene primers for SARS-CoV-2 detection (Supp Table 5 ). Overall, the primers had less than 80% homology with the vast majority of tested pathogen sequences. For any organisms where a primer hit >80% homology, only one of the primers (forward or reverse) had significant homology making an amplified product extremely unlikely. Overall, the results of this analysis predict no significant crossreactivity or microbial interference. We also assessed the potential impact of sequence variation in circulating strains that might lead to poor amplification. In the thousands of sequences deposited in GISAID (Shu and McCauley, 2017) , only one site in the priming region was observed to be polymorphic. The polymorphism (T30359C) was only observed in 106 of 6753 (<2%) sequences with coverage of this region. This variant overlaps the priming site of the LB primer but is not near a 3-prime end and is not anticipated to cause amplification failure.