Evaluation of Optimal Pooling Strategies for Mpox virus Genomic Surveillance Using the Illumina Viral Surveillance Panel on the iSeq100 Platform in Outbreak Settings

Genomic surveillance is essential 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 extend throughput without undermining enrichment yield, genome coverage, variant resolution or assembly quality, all of which are essential for genomic surveillance. Using Mpox virus (MPXV) positive clinical specimens collected during the 2024 outbreak in Burundi, we assessed the impact of pooling 3, 6, 9 and 15 samples per hybrid-capture reaction on viral enrichment yield, genome coverage, sequencing depth, variant resolution, detection sensitivity and assembly quality with the Illumina iSeq100 platform. The standard three-sample protocol yielded optimal performance across most parameters. However, pools of six samples maintained high genome coverage (≥90% at 10×), robust enrichment and reliable variant resolution, demonstrating that pooling up to six samples can preserve data quality for genomic surveillance and lineage tracking. In contrast, pools of nine or more showed marked performance losses, particularly for high Ct specimens, with reduced enrichment yield, genome completeness and variant sensitivity. Despite these limitations, viral identification remained robust even at higher pooling levels up to fifteen. Overall, our evaluation shows that multiplexing beyond three samples can balance cost-efficiency and genomic fidelity in resource-limited settings using the VSP/iSeq100 workflow. These insights support a tiered pooling strategy and provide a basis for adaptive genomic surveillance tailored to different outbreak response needs.