Gigante Crystal M, Weigand Michael R, Li Yu
National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.
National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.
Methods Mol Biol. 2025;2860:39-63. doi: 10.1007/978-1-0716-4160-6_4.
Poxviruses have exceptionally large genomes compared to most other viruses, which represent unique challenges to sequencing and assembly due to complex features such as repeat elements and low complexity sequences. The 2022 global mpox outbreak led to an unprecedented level of poxvirus sequencing as public health and research institutions faced with large sample numbers and demand for fast turnaround, merged NGS protocols designed for small RNA viruses with poxvirus expertise. Traditional manual assembly, checking, and editing of genomes was not feasible. Here, we present a protocol for metagenomic sequencing and orthopoxvirus genome assembly directly from DNA extracted from a patient lesion swab with no viral enrichment or host depletion. This sequencing approach is cost effective when using high throughput sequencing instruments and allows for detection of genomic insertions, deletions, and large rearrangement with confidence. We describe usage of two publicly available bioinformatic pipelines for genome assembly, quality control, annotation, and submission to sequence repositories.
与大多数其他病毒相比，痘病毒的基因组格外庞大，由于存在重复元件和低复杂性序列等复杂特征，这给测序和组装带来了独特挑战。2022年全球猴痘疫情导致痘病毒测序达到了前所未有的水平，因为公共卫生和研究机构面对大量样本以及快速周转的需求，将针对小RNA病毒设计的二代测序方案与痘病毒专业知识相结合。传统的手动基因组组装、检查和编辑并不可行。在此，我们展示了一种直接从患者皮损拭子提取的DNA进行宏基因组测序和正痘病毒基因组组装的方案，无需病毒富集或宿主去除。当使用高通量测序仪器时，这种测序方法具有成本效益，并且能够可靠地检测基因组插入、缺失和大型重排。我们描述了两种公开可用的生物信息学流程在基因组组装、质量控制、注释以及提交至序列数据库方面的用法。
