Development of Metagenomic RNA-Seq Standard Operating Procedure for Variant-agnostic Whole Genome Sequencing of SARS-CoV-2 isolates from Animal Samples

摘要 严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）在中国的遗传多样性研究 对非人类宿主的了解更少。虽然全基因组测序（WGS） 发现广泛应用于表征SARS-CoV-2分离株的遗传多样性， 在过去的两年里，目前的协议仅仅是为了确定在人类中传播的突变体， hosts.这种方法可能会错过新的生物学相关的SARS-CoV-2变异， 可能会出现这种病毒不断与不同的非人类宿主相互作用。拟议 因此，研究的重点是建立一个有效的标准操作程序 (SOP)直接测序和分析临床动物标本中的SARS-CoV-2， 宏基因组RNA-Seq方法。与最广泛使用的多重PCR靶向 扩增子测序方法（MTA-Seq），这种无偏的宏基因组RNA-Seq方法 忠实地覆盖了整个病毒基因组，并增加了发现新的和独特的病毒的几率。 可能不存在于人类群体中的变异。 我们将使用来自诊断病例的存档SARS-CoV-2阳性样本进行评估， 两种新的文库制备系统，其已知用于普遍减少宿主 背景（Zymo-Seq Ribo-Free Total RNA试剂盒）或用于增强病毒基因组 单引物等温扩增（SPIA）的扩增灵敏度（Revelo RNA-Seq Library Kit）。然后将在Illumina iSeq和MiSeq上对文库进行测序 平台我们将在测序的基础上比较两种WGS试剂盒的性能 深度、基因组覆盖率、测序均匀性和检测限。生物信息学分析 将在公共Galaxy服务器及其GalaxyTrakr实例中执行。经过充分验证的 本项目制定的SOP将提交给FDA Vet-LIRN，以分发给其他 实验室成员。这项工作完成后，将大大提高兽医的能力， LIRN实验室，并加强整个网络的合作。

Abstract The genetic diversity of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in non-human hosts is more poorly understood. Although whole-genome sequencing (WGS) has found extensive application in characterizing the genetic diversity of SARS-CoV-2 isolates over the past two years, current protocols are aimed solely at identifying mutants circulating in human hosts. This approach could potentially miss new biologically relevant SARS-CoV-2 variants that may emerge as this virus continuously interacts with disparate non-human hosts. The proposed research, therefore, focuses on the establishment of a validated standard operating procedure (SOP) to directly sequence and analyze SARS-CoV-2 in clinical animal specimens using the metagenomic RNA-Seq approach. Compared with the most widely used multiplex PCR targeted amplicon sequencing approach (MTA-Seq), this unbiased metagenomic RNA-Seq approach faithfully covers the entire viral genome and increases the odds of detecting new and unique variants that may not be present in human populations. We will use archived SARS-CoV-2 positive samples originated from diagnostic cases to evaluate two novel library preparation systems, which are known either for the universal reduction of host background (Zymo-Seq Ribo-Free Total RNA kit) or for enhancement of viral genome amplification sensitivity with single primer isothermal amplification (SPIA) (Revelo RNA-Seq Library Kit). The libraries will then be sequenced on both the Illumina iSeq and MiSeq platforms. We will compare the performance of both kits for WGS on the basis of sequencing depth, genome coverage, sequencing uniformity, and detection limits. Bioinformatic analyses will be performed in the public Galaxy server and its GalaxyTrakr instance. The fully validated SOP developed from this project will be submitted to FDA Vet-LIRN for distribution to other member laboratories. On completion, this work will significantly enhance the capabilities of Vet- LIRN laboratories and strengthen collaboration across the network.