Targeted Amplification and Genetic Sequencing of the Severe Acute Respiratory Syndrome Coronavirus 2 Surface Glycoprotein

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.

严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）刺突蛋白（spike protein）是一种高免疫原性且易于突变的蛋白，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点。SARS-CoV-2测序领域的绝大多数研究者采用基于铺层扩增子（tiling amplicon）的策略对全基因组进行测序。随着病毒不断进化，引物与模板的错配不可避免地导致扩增子扩增失败（amplicon drop-out）。由于刺突蛋白暴露于宿主抗体，该区域的突变发生速度最快，使得基因组中最具研究价值的区域出现扩增子扩增失败的问题。为缓解这一问题，我们开发了一种靶向扩增并测序S基因的方法。通过反复的计算机模拟（in silico）与体外（in vitro）实验测试，我们对20种不同的引物设计方案进行了评估，以筛选出最优的引物对及反应运行参数。筛选确定最优方案后，我们通过定期的计算机模拟分析，监测SARS-CoV-2进化过程中引物的保守性。尽管该引物设计方案是在贝塔（Beta）变异株流行期开发的，但截至2023年10月19日，经奥密克戎（Omicron）变异株演化后的引物保守性仍高于99%。为验证最终设计方案的有效性，我们选取321份匹配样本，将靶向S基因测序得到的数据与国家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S基因共识序列一致性高达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。