title: Modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis.
abstract: A major limitation for better understanding the role of the human gut virome in health and disease is the lack of validated methods that allow high throughput virome analysis. To overcome this, we evaluated the quantitative effect of homogenisation, centrifugation, filtration, chloroform treatment and random amplification on a mock-virome (containing nine highly diverse viruses) and a bacterial mock-community (containing four faecal bacterial species) using quantitative PCR and next-generation sequencing. This resulted in an optimised protocol that was able to recover all viruses present in the mock-virome and strongly alters the ratio of viral versus bacterial and 16S rRNA genetic material in favour of viruses (from 43.2% to 96.7% viral reads and from 47.6% to 0.19% bacterial reads). Furthermore, our study indicated that most of the currently used virome protocols, using small filter pores and/or stringent centrifugation conditions may have largely overlooked large viruses present in viromes. We propose NetoVIR (Novel enrichment technique of VIRomes), which allows for a fast, reproducible and high throughput sample preparation for viral metagenomics studies, introducing minimal bias. This procedure is optimised mainly for faecal samples, but with appropriate concentration steps can also be used for other sample types with lower initial viral loads.
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory of Viral Metagenomics, Rega Institute for Medical Research Leuven, Belgium.|KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory for Clinical Virology, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Nádia
authors.initials: N
authors.lastname: Conceição-Neto
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory of Viral Metagenomics, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Mark
authors.initials: M
authors.lastname: Zeller
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory of Viral Metagenomics, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Hanne
authors.initials: H
authors.lastname: Lefrère
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory of Viral Metagenomics, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: De Bruyn
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory of Viral Metagenomics, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Leen
authors.initials: L
authors.lastname: Beller
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory of Viral Metagenomics, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Ward
authors.initials: W
authors.lastname: Deboutte
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory of Viral Metagenomics, Rega Institute for Medical Research Leuven, Belgium.|KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory for Clinical Virology, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Claude Kwe
authors.initials: CK
authors.lastname: Yinda
authors.affiliation: KU Leuven - University of Leuven, Department of Biosystems, Laboratory of Gene Technology, Faculty of Bioscience Engineering, Belgium.
authors.forename: Rob
authors.initials: R
authors.lastname: Lavigne
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory for Clinical Virology, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Piet
authors.initials: P
authors.lastname: Maes
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory for Clinical Virology, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: Van Ranst
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory of Viral Metagenomics, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Heylen
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory of Viral Metagenomics, Rega Institute for Medical Research Leuven, Belgium.|KU Leuven - University of Leuven, Department of Microbiology and Immunology, Laboratory for Clinical Virology, Rega Institute for Medical Research Leuven, Belgium.
authors.forename: Jelle
authors.initials: J
authors.lastname: Matthijnssens
mesh_terms: D002498:Centrifugation / Q000379:methods; D005374:Filtration / Q000379:methods; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing; D054892:Metagenome; D056186:Metagenomics / Q000379:methods / Q000592:standards; D021141:Nucleic Acid Amplification Techniques; D014771:Virion / Q000302:isolation & purification; D014780:Viruses / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification