Efficient Whole-Genome Sequencing of Monkeypox Virus Using a Novel Nuclease-Multiple Displacement Amplification Enrichment Method

Monkeypox virus (MPXV) has a large double-stranded DNA genome (~ 200 kb), which presents challenges for whole-genome sequencing. Conventional enrichment methods have limitations, such as high cost or vulnerability to viral mutations. To address these issues, we developed a novel enrichment strategy combining nuclease treatment and multiple displacement amplification, along with terminal PCR to compensate for the reduced read depth at genome termini. When applied to 18 historical isolates, this method yielded more than 96% MPXV-specific reads, enabled complete genome assembly, and demonstrated reproducibility and robustness in phylogenetic analysis. Compared with hybridization capture- or PCR-based strategies, it is cost-effective and invulnerable to primer or probe mismatches due to viral mutations. While the method requires virus isolation, MPXV can be readily isolated from clinical samples. This strategy is broadly applicable to other poxviruses and double-stranded DNA viruses, supporting genomic surveillance, evolutionary studies, and outbreak preparedness in both research and public health settings.