A Comparison of Methods for the Optimal Recovery of the Human Fecal Virome
De Chiara, L.; Doughty, R.; Estevez-Gomez, N.; Gallego-Garcia, P.; Alvarino, P.; Diez-Martin, A.; Davila Pinon, P.; Treangen, T. J.; Cubiella, J.; Posada, D.
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Human virome studies are gaining attention as viruses are increasingly acknowledged as key modulators of microbial communities and human health. However, viral metagenomics presents distinct challenges, including the low abundance and diversity of viruses in biological samples, the lack of universal marker genes, and protocol-induced biases. Although various virome protocols have been benchmarked using viral particles or nucleic acids from mock communities, these often fail to replicate the complexity and heterogeneity of natural viromes. In this study, we systematically evaluated protocol modifications for the metagenomic analysis of human fecal samples, testing alternatives for viral enrichment, nucleic acid extraction, genome amplification, and library preparation. We assessed the impact of each modification on key inferences, including taxonomic and functional assignment, contig quality, viral diversity, and genome structure. Our results highlight important trade-offs between viral genome recovery and contamination removal, underscoring how methodological choices can shape virome composition. Based on our findings, we propose an optimized protocol that enhances the recovery of viral DNA and RNA genomes while minimizing contamination from non-viral sequences, providing a robust framework for future gut virome studies.
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