A Comparison of Methods for the Optimal Recovery of the Human Fecal Virome

ABSTRACT 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. Competing Interest Statement The authors have declared no competing interest. Funding Statement This project received funding from Fundacion Cientifica de la Asociacion Espanola contra el Cancer (PRYGN211425POSA), granted to D.P. R.D. is supported by a training fellowship from the Gulf Coast Consortia, on the NLM Training Program in Biomedical Informatics & Data Science (T15LM007093). T.J.T is supported in part by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (P01-AI152999, U19-AI144297) and the National Science Foundation (EF-2126387, IIS-2239114). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Individuals were recruited from Complexo Hospitalario Universitario de Ourense, and the samples were obtained from the Biobank of the Galicia Sur Health Research Institute (IISGS). Sample transfer was approved by the Ethics Committee for Clinical Research of Galicia (2021/134). Written informed consent was obtained from all participants, ensuring anonymity and adherence to ethical and clinical guidelines established by the Spanish Government and the Declaration of Helsinki. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability FASTQ files generated for this study are available in the BioProject repository with the accession number PRJNA1250239. All code and scripts used for bioinformatic processing, taxonomic assignment, assembly, richness and diversity analysis, and statistical analyses are available at: https://github.com/rdoughty10/GutVirome.