Using pangenome variation graphs to improve mutation detection in a large DNA virus.

Accurately quantifying viral genetic diversity is essential for understanding pathogen evolution, transmission and emergence. However, standard approaches that map sequencing reads to a single linear reference genome introduce substantial reference bias, particularly for samples that are divergent and recombinant or belong to rare lineages. Pangenome variation graphs (PVGs) mitigate this issue by representing multiple genomes within a unified graph structure, enabling read mapping across all observed and potential haplotypes. Despite this, PVGs have rarely been applied to viruses. Here, we address this gap by constructing and evaluating the first PVG for lumpy skin disease virus (LSDV), an emerging poxvirus of global importance. We generated PVGs of different sizes and mapped Illumina datasets using Giraffe, benchmarking performance against linear reference mapping with Minimap2. A minimal three-sample PVG containing one representative from each major lineage recovered 97% of known LSDV nucleotide diversity while reducing PVG size by >95% relative to a 121-sample PVG. PVG-based mapping detected more SNPs than linear mapping, including variants supported by read evidence that was not detected when reads were mapped to a single reference genome. Twenty-seven per cent of SNPs detected using PVG-based mapping could not be projected onto the linear reference coordinate system because they were on alternative paths absent from the reference, highlighting the impacts of reference bias. Notably, these new SNPs were at genes involved in host recognition and immune evasion and identified lineage-specific mutations that improved subclade phylogenetic structure. Our findings demonstrate that PVGs substantially enhance SNP discovery in LSDV, with direct implications for genomic surveillance, outbreak tracing and the detection of recombinant vaccine-related lineages in LSDV and other large DNA viruses.