Directional high-throughput sequencing of RNAs without gene-specific primers
Mäki, A., & Tiirola, M. (2018). Directional high-throughput sequencing of RNAs without gene-specific primers. BioTechniques, 65(4), 219-223. https://doi.org/10.2144/btn-2018-0082
© Authors, 2018
Ribosomal RNA analysis is a useful tool for characterization of microbial communities. However, the lack of broad-range primers has hampered the simultaneous analysis of eukaryotic and prokaryotic members by amplicon sequencing. We present a complete workfl ow for directional, primer-independent sequencing of size-selected small subunit ribosomal RNA fragments. The library preparation protocol includes gel extraction of the target RNA, ligation of an RNA oligo to the 5′-end of the target, and cDNA synthesis with a tailed random-hexamer primer and further barcoding. The sequencing results of a phytoplankton mock community showed a highly similar profi le to the biomass indicators. This method has universal potential for microbiome studies, and is compatible for the 5′-end sequencing of other RNA types with minimum library preparation costs.
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Lisätietoja rahoituksestaAM was supported by the European Research Council (ERC) through a grant awarded to MT under the European Union’s Seventh Framework Programme (FP/2007-2013, grant 615146). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. ...
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