Benchmarking Read-Based Virome Profilers for Human Virus Detection and Community Discovery

Na Rae Choi; Jung Hwa Park; Tae Sung Kim; Hee Sam Na

doi:10.4167/jbv.2025.55.4.350

All Issue

2025 Vol.55, Issue 4 Preview Page Next Page

Original Article

Benchmarking Read-Based Virome Profilers for Human Virus Detection and Community Discovery

31 December 2025. pp. 350-359

PDF XML

Abstract

Background: Shotgun metagenomics enables human virome profiling, yet read- based platforms differ in databases and mapping strategies, potentially altering biological conclusions, especially when goals diverge between human virus detection and broader virome discovery. Methods: We benchmarked Kraken2, FastViromeExplorer, and ViromeScan on whole-genome shotgun datasets from gut, nasal, oral, and vaginal sites. Reads were trimmed, human-depleted, and profiled; counts were normalized to CPM. We compared alpha diversity, beta diversity, and taxonomic composition. Results: Platform choice strongly affected viral composition. FastViromeExplorer produced the highest CPM and alpha diversity. Kraken2 was intermediate, and ViromeScan was lowest. Beta-diversity showed clear site clustering for all platforms, strongest with FastViromeExplorer, moderate with Kraken2, and weakest with ViromeScan. At the phylum level, Uroviricota dominated Kraken2 and FastViromeExplorer, whereas ViromeScan emphasized Nucleocytoviricota and Peploviricota. Genus and species calls were highly platform-dependent. FastViromeExplorer detected the most taxa overall, while ViromeScan preferentially reported eukaryotic and human viruses. Conclusions: Read-based profilers yield divergent virome portraits driven by database scope and mapping stringency. For clinical human-virus detection, curated human and eukaryotic references with coverage-based confirmation are essential. Our results provide practical guidance for aligning pipelines to study goals and underscore the need to report parameters, database versions, and cross-platform validations when interpreting the human virome.

Keywords

Virome profile

Benchmark

Next generation sequencing

Whole genome sequencing

References

Lathakumari RH, Vajravelu LK, Gopinathan A, Vimala PB, Panneerselvam V, Ravi SSS, et al. The gut virome and human health: From diversity to personalized medicine. Eng Microbiol. 2025;5(1):100191.

10.1016/j.engmic.2025.10019140538711PMC12173812

Khokhar RK, Nashwan AJ. Gut virome and its emerging role in inflammatory bowel disease. World J Methodol. 2025;15(3):100534.

10.5662/wjm.v15.i3.10053440881220PMC11948198

Virgin HW. The virome in mammalian physiology and disease. Cell. 2014;157(1):142-150.

10.1016/j.cell.2014.02.03224679532PMC3977141

Norman JM, Handley SA, Baldridge MT, Droit L, Liu CY, Keller BC, et al. Disease-specific alterations in the enteric virome in inflammatory bowel disease. Cell. 2015;160(3):447-460.

10.1016/j.cell.2015.01.00225619688PMC4312520

Sulek K. [Nobel prizes for John F. Enders, Frederick Ch, Robbins and Thomas H. Weller in 1954 for discovery of the possibility of growing poliomyelitis virus on various tissue media]. Wiad Lek. 1968;21(24):2301-2303.

Bibby K. Metagenomic identification of viral pathogens. Trends Biotechnol. 2013;31(5):275-279.

10.1016/j.tibtech.2013.01.016

Hall RJ, Draper JL, Nielsen FG, Dutilh BE. Beyond research: a primer for considerations on using viral metagenomics in the field and clinic. Front Microbiol. 2015;6:224.

10.3389/fmicb.2015.0022425859244PMC4373370

Roux S, Enault F, Hurwitz BL, Sullivan MB. VirSorter: mining viral signal from microbial genomic data. PeerJ. 2015;3:e985.

10.7717/peerj.98526038737PMC4451026

Ren J, Ahlgren NA, Lu YY, Fuhrman JA, Sun F. VirFinder: a novel k-mer based tool for identifying viral sequences from assembled metagenomic data. Microbiome. 2017;5(1):69.

10.1186/s40168-017-0283-528683828PMC5501583

Roux S, Tournayre J, Mahul A, Debroas D, Enault F. Metavir 2: new tools for viral metagenome comparison and assembled virome analysis. BMC bioinformatics. 2014;15:76.

10.1186/1471-2105-15-7624646187PMC4002922

Yang C, Chowdhury D, Zhang Z, Cheung WK, Lu A, Bian Z, et al. A review of computational tools for generating metagenome-assembled genomes from metagenomic sequencing data. Comput Struct Biotechnol J. 2021;19:6301-6314.

10.1016/j.csbj.2021.11.02834900140PMC8640167

Wood DE, Lu J, Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biol. 2019;20(1):257.

10.1186/s13059-019-1891-031779668PMC6883579

Rampelli S, Soverini M, Turroni S, Quercia S, Biagi E, Brigidi P, et al. ViromeScan: a new tool for metagenomic viral community profiling. BMC genomics. 2016;17:165.

10.1186/s12864-016-2446-326932765PMC4774116

Tithi SS, Aylward FO, Jensen RV, Zhang L. FastViromeExplorer-Novel: Recovering Draft Genomes of Novel Viruses and Phages in Metagenomic Data. J Comput Biol. 2023;30(4):391-408.

10.1089/cmb.2022.0397

Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA, et al. The NIH Human Microbiome Project. Genome Res. 2009;19(12) :2317-2323.

10.1101/gr.096651.10919819907PMC2792171

Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, et al. Metagenomic biomarker discovery and explanation. Genome Biol. 2011;12(6):R60.

10.1186/gb-2011-12-6-r6021702898PMC3218848

Ghurye JS, Cepeda-Espinoza V, Pop M. Metagenomic Assembly: Overview, Challenges and Applications. Yale J Biol Med. 2016;89(3):353-362.

Li D, Liu CM, Luo R, Sadakane K, Lam TW. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 2015;31(10):1674-1676.

10.1093/bioinformatics/btv033

Sczyrba A, Hofmann P, Belmann P, Koslicki D, Janssen S, Dröge J, et al. Critical Assessment of Metagenome Interpretation-a benchmark of metagenomics software. Nat Methods. 2017;14(11):1063-1071.

10.1038/nmeth.445828967888PMC5903868

Liu Y, Demina TA, Roux S, Aiewsakun P, Kazlauskas D, Simmonds P, et al. Diversity, taxonomy, and evolution of archaeal viruses of the class Caudoviricetes. PLoS Biol. 2021;19(11):e3001442.

10.1371/journal.pbio.300144234752450PMC8651126

Aylward FO, Moniruzzaman M, Ha AD, Koonin EV. A phylogenomic framework for charting the diversity and evolution of giant viruses. PLoS Biol. 2021;19(10):e3001430.

10.1371/journal.pbio.300143034705818PMC8575486

Colson P, De Lamballerie X, Yutin N, Asgari S, Bigot Y, Bideshi DK, et al. “Megavirales”, a proposed new order for eukaryotic nucleocytoplasmic large DNA viruses. Arch Virol. 2013;158(12):2517-2521.

10.1007/s00705-013-1768-623812617PMC4066373

Dotto-Maurel A, Arzul I, Morga B, Chevignon G. Herpesviruses: overview of systematics, genomic complexity and life cycle. Virol J. 2025;22(1):155.

10.1186/s12985-025-02779-740399963PMC12096621

Cossart YE. The rise and fall of infectious diseases: Australian perspectives, 1914-2014. Med J Aust. 2014;201(1 Suppl):S11-4.

10.5694/mja14.0011225047768PMC7168456

Van Doorslaer K, Chen Z, Bernard HU, Chan PKS, DeSalle R, Dillner J, et al. ICTV Virus Taxonomy Profile: Papillomaviridae. J Gen Virol. 2018;99(8):989-990.

10.1099/jgv.0.00110529927370PMC6171710

Kim M, Parrish RC 2nd, Tisza MJ, Shah VS, Tran T, Ross M, et al. Host DNA depletion on frozen human respiratory samples enables successful metagenomic sequencing for microbiome studies. Commun Biol. 2024;7(1):1590.

10.1038/s42003-024-07290-339609616PMC11604929

Information

Publisher :The Korean Society for Microbiology and The Korean Society of Virology
Publisher(Ko) :대한미생물학회‧대한바이러스학회
Journal Title :JOURNAL OF BACTERIOLOGY AND VIROLOGY
Volume : 55
No :4
Pages :350-359
Received Date : 2025-09-30
Revised Date : 2025-11-25
Accepted Date : 2025-12-03
DOI :https://doi.org/10.4167/jbv.2025.55.4.350

JOURNAL OF BACTERIOLOGY AND VIROLOGY ISSN:1598-2467(Print) 2093-0429(Online)

All Issue