Targeted S-gene Sequencing Supplemental Material

The SARS-CoV-2 spike protein is a highly immunogenic and mutable protein that is the target of vaccine prevention and antibody therapeutics. This makes the encoding S-gene an important sequencing target. The SARS-CoV-2 sequencing community overwhelmingly adopted tiling amplicon-based strategies for sequencing the entire genome. As the virus evolved, primer mismatches inevitably led to amplicon drop-out. Given the exposure of the spike protein to host antibodies, mutation occurred here most rapidly, leading to amplicon failure over the most insightful region of the genome. To mitigate this, we developed a targeted method to amplify and sequence the S-gene. We evaluated 20 distinct primer designs through iterative in silico and in vitro testing to select the optimal primer pairs and run conditions. Once selected, periodic in silico analysis monitor primer conservation as SARS-CoV-2 evolves. Despite being designed during the Beta wave, the selected primers remain > 99% conserved through Omicron as of 2023-10-19. To validate the final design, we compared targeted S-gene data to National SARS-CoV-2 Strain Surveillance whole-genome data for 321 matching samples. Consensus sequences for the two methods were highly identical (99.998%) across the S-gene. This method can serve as a complement to whole-genome surveillance or be leveraged where only S-gene sequencing is of interest.