Researchers from Université de Tours, France 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, the researchers also found a gap between the two sequencing approaches. Collectively, this 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.
Next-generation sequencing data considered alone are limited for assessing the absolute copy number of microRNA transcripts
Stik G, Muylkens B, Coupeau D, Laurent S, Dambrine G, Mélanie M, Béatrice CW, Pfeffer S, Rasschaert D. (2014) Small RNA cloning and sequencing strategy affects host and viral microRNA expression signatures. J Biotechnol [Epub ahead of print]. [abstract]