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

In this study, we developed a novel enrichment method for whole-genome sequencing of Monkeypox virus (MPXV), which has a large double-stranded DNA genome that poses technical challenges. The method combines nuclease treatment and multiple displacement amplification (MDA), supplemented with terminal PCR to compensate for reduced read depth at the genome termini. We demonstrated that this approach yielded over 96% MPXV-specific reads from 18 historical MPXV strains, enabling complete genome assembly. Compared to conventional hybridization capture- or PCR-based strategies, this method is more cost-effective and is less vulnerable to primer or probe mismatches due to viral mutations.