Impact of phage enrichment on the observed infant gut phageome

The human gut microbiota, particularly during infancy, plays a pivotal role in shaping long-term health outcomes. While research on the bacterial microbiota has advanced rapidly, the infant gut virome-dominated by bacteriophages-remains underexplored due to technical challenges in viral DNA detection and recovery. To address this, we optimized a polyethylene glycol (PEG)-based protocol for phage DNA enrichment tailored to the constraints of infant fecal samples, focusing on maximizing viral yield from minimal input material. We validated the optimized protocol on fecal samples from 41 infants at 1, 6, and 12 months of age and assessed the impact of phage enrichment on the observed gut phageome. The results demonstrate that the optimized protocol improves viral DNA recovery and significantly alters the observed virome composition, especially in older infants. Without appropriate enrichment, key features of the gut virome may be underrepresented or missed entirely. These findings underscore the importance of protocol optimization in virome studies and provide a scalable, cost-effective method for robust infant gut virome profiling.IMPORTANCEUnderstanding the viral component of the infant gut microbiome is essential for uncovering its role in early-life health, yet technical limitations have hindered its study. This work presents a systematically optimized and validated protocol for enriching viral DNA from infant stool samples, designed specifically for low-input material typical of early life. By adapting polyethylene glycol-based precipitation methods, we achieved consistent and scalable recovery of viral DNA across infants of different ages. Application of this protocol revealed key age- and delivery mode-specific differences in phage diversity and replication strategies that were undetectable using standard approaches. Our findings demonstrate that careful protocol optimization is critical for accurate virome profiling in infants and offer a practical solution to overcome longstanding methodological challenges in the field.