title: Small RNA cloning and sequencing strategy affects host and viral microRNA expression signatures.
abstract: The establishment of the microRNA (miRNA) expression signatures is the basic element to investigate the role played by these regulatory molecules in the biology of an organism. Marek's disease virus 1 (MDV-1) is an avian herpesvirus that naturally infects chicken and induces T cells lymphomas. During latency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include three miRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1 infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequencies obtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effect of small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNA samples collected during MDV-1 infection of chicken at different steps of the oncoviral pathogenesis. Qualitative and quantitative variations were found in the data, depending on the strategy used. One of the mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed the highest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencing approach was used. Its frequency was 100 times less abundant when determined through the deep sequencing approach. Northern blot analysis showed a better correlation with the miRNA frequencies found by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also found a gap between the two sequencing approaches. Collectively, our study indicates that next-generation sequencing data considered alone are limited for assessing the absolute copy number of transcripts. Thus, the quantification of small RNA should be addressed by compiling data obtained by using different techniques such as microarrays, qRT-PCR and NB analysis in support of high throughput sequencing data. These observations should be considered when miRNA variations are studied prior addressing functional studies.
authors.affiliation: Transcription, Lymphome Viro-Induit, UMR7261, Université de Tours, UFR Sciences et Techniques, Parc Grandmont, 37200 Tours, France.
authors.forename: Grégoire
authors.initials: G
authors.lastname: Stik
authors.affiliation: Unité de Recherche Vétérinaire Intégrée, Narilis, Faculté des sciences, Université de Namur, rue de Bruxelles 61, B-5000 Namur, Belgium.
authors.forename: Benoît
authors.initials: B
authors.lastname: Muylkens
authors.affiliation: Transcription, Lymphome Viro-Induit, UMR7261, Université de Tours, UFR Sciences et Techniques, Parc Grandmont, 37200 Tours, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Coupeau
authors.affiliation: Transcription, Lymphome Viro-Induit, UMR7261, Université de Tours, UFR Sciences et Techniques, Parc Grandmont, 37200 Tours, France; INRA - Département de Santé Animale, Centre de recherches de Tours, 37380 Nouzilly, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Laurent
authors.affiliation: Transcription, Lymphome Viro-Induit, UMR7261, Université de Tours, UFR Sciences et Techniques, Parc Grandmont, 37200 Tours, France; INRA - Département de Santé Animale, Centre de recherches de Tours, 37380 Nouzilly, France.
authors.forename: Ginette
authors.initials: G
authors.lastname: Dambrine
authors.affiliation: Institut de Biologie Moléculaire et Cellulaire, Architecture et Réactivité de l'ARN, CNRS, UPR9002, 15 Rue René Descartes, 67084 Strasbourg, France.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Messmer
authors.affiliation: Institut de Biologie Moléculaire et Cellulaire, Architecture et Réactivité de l'ARN, CNRS, UPR9002, 15 Rue René Descartes, 67084 Strasbourg, France.
authors.forename: Béatrice
authors.initials: B
authors.lastname: Chane-Woon-Ming
authors.affiliation: Institut de Biologie Moléculaire et Cellulaire, Architecture et Réactivité de l'ARN, CNRS, UPR9002, 15 Rue René Descartes, 67084 Strasbourg, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Pfeffer
authors.affiliation: Transcription, Lymphome Viro-Induit, UMR7261, Université de Tours, UFR Sciences et Techniques, Parc Grandmont, 37200 Tours, France. Electronic address: denis.rasschaert@univ-tours.fr.
authors.forename: Denis
authors.initials: D
authors.lastname: Rasschaert
mesh_terms: D000818:Animals; D002645:Chickens / Q000235:genetics / Q000821:virology; D008381:Herpesvirus 2, Gallid / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D008380:Marek Disease / Q000235:genetics / Q000821:virology; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis*; D059467:Transcriptome
chemical_list: D035683:MicroRNAs; D012367:RNA, Viral
keywords: Marek's disease virus; MicroRNA; Small RNA cloning and sequencing; Viral infection