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 <i>in silico</i> and <i>in vitro</i> testing to select the optimal primer pairs and run conditions. Once selected, periodic <i>in silico</i> analysis monitor primer conservation as SARS-CoV-2 evolves. Despite being designed during the Beta wave, the selected primers remain &gt; 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.

SARS-CoV-2刺突蛋白（spike protein）是一种高免疫原性且易变异的蛋白，同时也是疫苗预防与抗体治疗的作用靶点。这使得编码该蛋白的S基因成为重要的测序靶标。新冠病毒测序领域绝大多数采用基于铺瓦式扩增子的策略对全基因组进行测序。随着病毒不断演化，引物错配不可避免地会导致扩增子脱落。由于刺突蛋白暴露于宿主抗体环境中，其突变速率最快，使得基因组中最具科研价值的区域出现扩增子失效问题。为缓解这一难题，我们开发了一种靶向扩增并测序S基因的方法。我们通过迭代的计算机（in silico）与体外（in vitro）测试评估了20种不同的引物设计方案，以筛选最优引物对及测序运行条件。完成引物筛选后，我们会定期通过计算机分析监测引物在SARS-CoV-2演化过程中的保守性。尽管该引物设计方案是在贝塔毒株流行期间完成的，但截至2023年10月19日，所选引物在奥密克戎毒株传播阶段的保守性仍高于99%。为验证最终设计方案的有效性，我们将靶向S基因测序得到的数据与321份匹配样本的国家SARS-CoV-2毒株监测全基因组数据进行了对比。两种方法得到的共识序列在S基因区域的一致性高达99.998%。该方法可作为全基因组监测的补充手段，或在仅需开展S基因测序的研究场景中应用。