Single-Nucleotide Sequencing of N6-methyladenosine in SARS-CoV2 RNA (COVID-19)

了解SARS-CoV-2病毒感染所涉及的生物过程是有效治疗和预防新冠肺炎爆发和未来潜在的冠状病毒大流行所必需的。阐明病毒复制周期及其进化的生物学始于其基因组RNA的测序。这有助于识别涉及的关键蛋白质和随着新冠肺炎大流行的发展而发生的突变。病毒的RNA序列只讲述了故事的一部分-最近有证据表明，病毒RNA在受感染的宿主细胞内的甲基化可以改变病毒蛋白质的合成和表达，并使病毒能够“伪装”自己以逃避宿主的免疫系统。确定这些甲基化位点在RNA中的位置对于指导未来应对COVID大流行的研究至关重要；然而，目前缺乏以高通量方式在单核苷酸水平上定量识别这些位置的方法。建议的测序方法将实施与现有的高通量测序系统兼容的高度选择性的化学过程，并容易识别腺苷N6位甲基化的存在和水平--腺苷是最普遍的甲基化形式。为了实现这个项目的目标，我们正在与玛格丽特公主癌症中心及其位于多伦多的生物库合作，后者已经开发了一个新冠肺炎+患者样本库。该团队共同提供最先进的基因组学专业知识和设施，以开发拟议的技术，并绘制受感染人类样本中SARS-CoV-2RNA的基因组甲基化图景。我们为期一年的研究计划是可行的，预计将导致开发一个强大的测序平台，用于基因组中心，以促进人类冠状病毒感染中RNA甲基化的生物学研究。

Understanding the biological processes involved in viral infection of SARS-CoV-2 is required to effectively treat and prevent COVID-19 outbreaks and potential future coronavirus pandemics. Elucidating the biology of virus replication cycles and its evolution begins with sequencing of its genomic RNA. This facilitate the identification of key proteins involved and mutations as the COVID-19 pandemic develops. The RNA sequence of a virus only tells part of the story - methylation of viral RNA within infected host cells has been recently shown to alter synthesis and expression of viral proteins and enable viruses to "disguise" themselves to evade the host's immune system. Determining the location of these methylation sites in RNA is critical to guiding future research to tackle COVID pandemics; however, current methods to identify these locations quantitatively and at the single-nucleotides level in a high-throughput manner is lacking. The proposed sequencing method will implement a highly-selective chemical process that is compatible with currently existing high-throughput sequencing systems and readily identifies the presence and level of methylation at the N6 -position of adenosine - the most prevalent methylation form. To achieve the goals of this project, we are partnering with Princess Margaret Cancer Centre and its BioBank in Toronto which has developed a repository of COVID-19+ patient samples. Together, the team provides state-of-the-art genomics expertise and facilities to develop the proposed technology and map the genomic methylation landscape of SARS-CoV-2 RNA in infected human samples. Our one-year research plan is feasible and expected to lead to the development of a robust sequencing platform to be used in the genomic centres to facilitate research in the biology of RNA methylation in coronavirus infection in humans.