Evaluation of optimal pooling strategies for Mpox virus genomic surveillance using the Illumina Viral Surveillance Panel on the iSeq100 platform in outbreak settings.

Introduction: Genomic surveillance is critical for outbreak control and pathogen intelligence, yet protocols such as the Illumina Viral Surveillance Panel (VSP) remain under-evaluated in low-resource and mobile laboratory settings. A key operational question is whether sample pooling can increase throughput without compromising enrichment yield, genome coverage, variant resolution or assembly quality. Methods: We conducted an empirical evaluation using Mpox virus (MPXV)-positive clinical specimens collected during the 2024 outbreak in Burundi. Hybrid-capture reactions were performed with pooled sets of 3, 6, 9 and 15 samples, sequenced on the Illumina iSeq100 platform. We assessed viral enrichment yield, genome coverage, sequencing depth, variant resolution, detection sensitivity and assembly quality across pooling levels. Results: The standard three-sample protocol yielded optimal performance across most parameters. Pools of six samples maintained high genome coverage (≥90% at 10×), robust enrichment and reliable variant resolution, indicating that pooling up to six samples preserves data quality for genomic surveillance. Pools of nine or more showed marked performance losses, particularly for high-Ct specimens, with reduced enrichment yield, genome completeness and variant sensitivity. Nonetheless, viral identification remained feasible even at pooling levels up to fifteen. Conclusions: Multiplexing beyond three samples can balance cost-efficiency and genomic fidelity in resource-limited settings using the VSP/iSeq100 workflow. These findings support a tiered pooling strategy and provide a practical basis for adaptive genomic surveillance tailored to outbreak response needs.