Human outbreak detection and best practice MPXV analysis and interpretation with squirrel.

High numbers of reported mpox cases and recent identification of multiple sustained human outbreaks of mpox virus (MPXV) have highlighted the need for robust, best-practice genomic surveillance tools. In light of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, many labs across the globe developed the capacity to do virus genome sequencing; however, MPXV presents additional analytical challenges due to its large genome size, tracts of low-complexity or repeat regions, genetically distinct clades, and the need to perform bespoke apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3)-mutation reconstruction. We present squirrel (Some Quick Reconstruction to Resolve Evolutionary Links), an open source bioinformatic tool that can perform clade-aware alignment, mutation quality assessment, phylogenetic inference, and automated APOBEC3-mutation classification on branches of the phylogeny. Squirrel can be run on the command line or launched through the EPI2ME graphical user interface through the squirrel-nf workflow, enabling robust analysis without need for the command line. With the interactive output report produced and publication-ready APOBEC3-reconstruction visualization, squirrel enables researchers to distinguish between zoonotic and sustained human outbreaks and help accurately inform public health responses.