title: Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes.
abstract: BACKGROUND
  Metagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles.
  
  
  RESULTS
  We evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7-15% of all reads were classified as "unknown", depending on the random amplification method.
  
  
  CONCLUSIONS
  The protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses.
authors.affiliation: Unité de Recherche sur les Maladies Infectieuses Tropicales Emergentes (URMITE) UM63, CNRS 7278, IRD 198, INSERM 1095, Aix-Marseille Université, Marseille, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Temmam
authors.affiliation: Unité de Recherche sur les Maladies Infectieuses Tropicales Emergentes (URMITE) UM63, CNRS 7278, IRD 198, INSERM 1095, Aix-Marseille Université, Marseille, France.
authors.forename: Sonia
authors.initials: S
authors.lastname: Monteil-Bouchard
authors.affiliation: Unité de Recherche sur les Maladies Infectieuses Tropicales Emergentes (URMITE) UM63, CNRS 7278, IRD 198, INSERM 1095, Aix-Marseille Université, Marseille, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Robert
authors.affiliation: Centre de Recherche et de Veille sur les maladies émergentes dans l'Océan Indien (CRVOI), IRD La Réunion, Plateforme de Recherche CYROI, La Réunion, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Pascalis
authors.affiliation: Unité de Recherche sur les Maladies Infectieuses Tropicales Emergentes (URMITE) UM63, CNRS 7278, IRD 198, INSERM 1095, Aix-Marseille Université, Marseille, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Michelle
authors.affiliation: Unité de Recherche sur les Maladies Infectieuses Tropicales Emergentes (URMITE) UM63, CNRS 7278, IRD 198, INSERM 1095, Aix-Marseille Université, Marseille, France.
authors.forename: Priscilla
authors.initials: P
authors.lastname: Jardot
authors.affiliation: Emergence des Pathologies Virales (EPV), IRD 190, EHESP, Aix-Marseille Université, Marseille, France.
authors.forename: Rémi
authors.initials: R
authors.lastname: Charrel
authors.affiliation: Unité de Recherche sur les Maladies Infectieuses Tropicales Emergentes (URMITE) UM63, CNRS 7278, IRD 198, INSERM 1095, Aix-Marseille Université, Marseille, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Raoult
authors.affiliation: Unité de Recherche sur les Maladies Infectieuses Tropicales Emergentes (URMITE) UM63, CNRS 7278, IRD 198, INSERM 1095, Aix-Marseille Université, Marseille, France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Desnues
mesh_terms: D056186:Metagenomics; D012367:RNA, Viral / Q000235:genetics*
chemical_list: D012367:RNA, Viral