A comparison of methods for the optimal recovery of the human fecal virome

Human virome research is gaining increasing attention as viruses are recognized as critical modulators of microbial communities and human health. Viral metagenomics, however, faces unique challenges, including the low abundance and diversity of viruses in biological samples, the absence of universal marker genes, and biases introduced by experimental protocols. While various virome protocols have been benchmarked using viral particles or nucleic acids from mock communities, these approaches often fail to capture the complexity and heterogeneity of natural viromes. In this study, we systematically evaluated modifications to key methodological steps in the metagenomic analysis of human fecal samples, including viral enrichment, nucleic acid extraction, genome amplification, and library preparation. Using gold-standard bioinformatic approaches on sequencing datasets generated after amplification, we assessed the impact of these modifications on relative viral taxonomic assignment, contig quality, richness, diversity, and inferred genome structure. Our findings reveal striking trade-offs between recovery of viral genomes and retention of nonviral sequences, demonstrating how methodological choices can shape the inferred virome composition. Based on these observations, we propose an optimized protocol that enhances viral genome recovery while reducing contamination from nonviral sequences. This refined workflow provides a more robust and reliable framework for gut virome studies, paving the way for a deeper exploration of the role of viruses in human health and microbial ecosystems.