Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named as 2019-nCoV) is the cause of novel coronavirus disease termed COVID-19. The virus originated from Wuhan, China and rapidly spreaded around the world causing a global pandemic (Worldometers.info, 2020). Sequencing of the virus showed that its single-stranded RNA genome is ~30 kb in size (Chan et al., 2020; Lu et al., 2020; Wu et al., 2020; Zhou et al., 2020). Availability of the viral sequence early in the outbreak helped the development of several polymerase chain reaction (PCR) detection protocols that have been instrumental in the diagnosis of the disease from patients samples (WHO, 2020). However, genetic variability in the viral genome during natural evolution poses a potential risk of mismatches between the diagnostic assays and the template that can result in false-negative results (Whiley and Sloots, 2005; Chow et al., 2011). Sequences of SARS-CoV-2 viruses isolated from around the world are being deposited in the sequence databases and mutations have been identified in the genomes of the circulating viruses (Ugurel et al., 2020).

We performed an extensive evaluation of published diagnostic PCR assays, including those recommended by the World Health Organization (WHO), based on evaluation of sequence variation in the primer/probe binding regions using more than 17,000 publicly available viral sequences (Khan and Cheung, 2020). Another concurrent publication reported mutations in primer/probe binding regions using 1825 sequences but a detailed sequence tracing protocol was not provided (Osorio and Correia-Neves, 2020). This step-by-step protocol outlines a bioinformatics pipeline that uses freely available open-source software programs. The pipeline can be performed on a regular desktop computer without any need for special hardware and does not require extensive computational skills. The provision of a ready-to-use Multiple Sequence Alignment (MSA) file through Open Science Framework (OSF) makes it an even more intuitive task. Inclusivity analysis through verification of in silico nucleotide identity match is one of the regulatory requirements for approval of COVID-19 diagnostic assays (Commission-Services, 2020; FDA, 2020; Health-Canada, 2020). The protocol can also be applied to other molecular diagnostic assays of SARS-CoV-2 including point-of-care CRISPR-based diagnostic assays under development (Tsang and LaManna, 2020).