Development and validation of a new mpox virus sequencing and bioinformatic analysis pipeline

Sequencing and bioinformatic analysis of mpox virus (MPXV) remain challenging in resource-limited settings. We developed and validated a PCR-based sequencing assay that targets a 12.5 kilobase (kb) region that is phylogenetically representative of the whole ∼ 200 kb MPXV genome. We combined this sequencing assay with a lightweight, downloadable, on-and-off-grid-bioinformatics pipeline for rapid phylogenetic analysis. Our findings demonstrate that this simplified sequencing method, and the associated bioinformatics pipeline accurately distinguished clades, subclades, and clusters of MPXV. Therefore, this assay will provide rapid sequence information for understanding transmission patterns and sources of outbreaks in resource-limited settings. In addition, this assay provides a unique opportunity to decentralize mpox molecular surveillance capacities that are needed to contain the ongoing outbreak.