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

制定宏基因组 RNA-Seq 标准操作程序，对动物样本中的 SARS-CoV-2 分离株进行变异不可知的全基因组测序

• 批准号: 10665686
• 负责人: Solomon Olawole Odemuyiwa
• 金额: $ 6.25万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10665686
• 关键词: Development; Metagenomic; RNA; Seq; Standard

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.

抽象的 严重急性呼吸综合征冠状病毒-2 (SARS-CoV-2) 的遗传多样性 对非人类宿主的了解则更甚少。尽管全基因组测序（WGS） 发现广泛应用于表征 SARS-CoV-2 分离株的遗传多样性 过去两年，目前的协议仅旨在识别人类中传播的突变体 主机。这种方法可能会错过新的生物学相关的 SARS-CoV-2 变体，这些变体 当这种病毒不断与不同的非人类宿主相互作用时，可能会出现这种情况。拟议的 因此，研究的重点是建立经过验证的标准操作程序 (SOP) 使用以下方法直接对临床动物标本中的 SARS-CoV-2 进行测序和分析 宏基因组 RNA-Seq 方法。与最广泛使用的多重PCR靶向相比 扩增子测序方法 (MTA-Seq)，这种无偏见的宏基因组 RNA-Seq 方法 忠实地覆盖整个病毒基因组，增加检测新的和独特的病毒的几率 人类中可能不存在的变异。 我们将使用源自诊断病例的存档 SARS-CoV-2 阳性样本来评估 两种新颖的文库制备系统，它们以普遍减少宿主而闻名 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 库套件）。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、基因组覆盖度、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。经充分验证 根据该项目制定的 SOP 将提交给 FDA Vet-LIRN，以分发给其他机构 成员实验室。完成后，这项工作将显着增强兽医的能力 LIRN 实验室并加强整个网络的协作。