RNA-Sequencing of Saliva Samples from COVID-Infected and Vaccinated Subjects

SARS-CoV-2 virus is known to infect the oral cavity and can be readily detected using PCR-based testing. In this study, we examined the host transcriptomic response to PCR-confirmed SARS-CoV-2 virus infection, vaccination against SARS-CoV-2, or breakthrough infection following vaccination using RNA-sequencing. The identification of the viral variant for all samples was obtained using full viral genome sequencing. Approximately equal numbers of males and females were used for every major variant lineage. Results indicate strong anti-viral responses in each case, with some differences due to variant strain and vaccination history, as well as age and sex. Human subjects participating in COVID-19 surveillance testing provided consent and submitted a saliva sample using a swab-based collection device. PCR testing was performed and SARS-CoV-2 genome sequencing completed on all positive subjects. Vaccine histories were also obtained. RNA was purified and used to create strand-specific libraries for next generation sequencing (RNA-Seq) with single-end reads. Resulting reads were mapped and quantified at the transcript level using Salmon, and filtered to remove features with less than 1 observed count in fewer than 80% of the samples. Normalization was applied using TPM + 1, followed by quantile normalization and log2 transformation.

已知严重急性呼吸综合征冠状病毒2（SARS-CoV-2）可感染口腔，且可通过基于聚合酶链反应（PCR）的检测方法便捷检出。本研究利用RNA测序（RNA-sequencing）技术，分析经PCR确认的SARS-CoV-2感染、新冠疫苗接种，或接种后突破感染的宿主转录组应答反应。所有样本的病毒变异株鉴定均通过全病毒基因组测序完成。各主要变异株谱系的受试样本中，男性与女性的数量大致相当。研究结果显示，各类研究对象均呈现强烈的抗病毒应答，且应答差异受变异毒株、疫苗接种史、年龄及性别影响。参与新型冠状病毒肺炎（COVID-19）监测检测的受试者已签署知情同意书，并通过拭子采集装置提交唾液样本。对所有新冠病毒检测呈阳性的受试者完成PCR检测及SARS-CoV-2基因组测序，同时收集其疫苗接种史。提取并纯化RNA后，构建链特异性文库，用于开展单端读长的下一代测序（RNA-Seq）。利用Salmon软件将所得读段比对至转录组水平并进行定量，随后过滤掉在低于80%的样本中观测计数小于1的转录本特征。采用每百万转录本数（TPM）+1进行标准化处理，后续依次进行分位数标准化及log₂转换。