Small RNA cloning and sequencing strategy affects host and viralmicroRNA expression signatures

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Stik, G. | Muylkens, Benoît | Coupeau, D. | Laurent, Sylvie | Ginette, DAMBRINE | Messmer, Mélanie | Chane-Woon-Ming, Béatrice | Pfeffer, Sébastien | Rasschaert, Denis

Edité par HAL CCSD ; Elsevier

International audience. tThe establishment of the microRNA (miRNA) expression signatures is the basic element to investigatethe 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. Duringlatency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include threemiRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequenciesobtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effectof small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNAsamples 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 ofthe mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed thehighest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencingapproach was used. Its frequency was 100 times less abundant when determined through the deepsequencing approach. Northern blot analysis showed a better correlation with the miRNA frequenciesfound by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also founda gap between the two sequencing approaches. Collectively, our study indicates that next-generationsequencing 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 differenttechniques 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 functionalstudies.

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