Evaluation of the Sequence Variability within the PCR Primer/Probe Target Regions of the SARS-CoV-2 Genome

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named 2019-nCoV) is responsible for the recent coronavirus disease (COVID-19) pandemic, and polymerase chain reaction (PCR) is the current standard method for diagnosis from patient samples. As PCR assays are prone to sequence mismatches due to mutations in the viral genome, it is important to verify the genomic variability at primer/probe binding regions periodically. This step-by-step protocol describes a bioinformatics approach for an extensive evaluation of the sequence variability within the primer/probe target regions of the SARS-CoV-2 genome. The protocol can be applied to any molecular diagnostic assay of choice using freely available software programs and the ready-to-use multiple sequence alignment (MSA) file provided. Graphic abstract Overview of the sequence tracing protocol. The figure was created using the Library of Science and Medical Illustrations from somersault18:24 licensed under a CC BY-NC-SA 4.0 license (https://creativecommons.org/licenses/by-nc-sa/4.0/). Video abstract: https://youtu.be/M1lV1liWE9k.