Orthopoxvirus Genome Sequencing, Assembly, and Analysis

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.