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
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BioTechniquesDate
2018Copyright
© 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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Future ScienceISSN Search the Publication Forum
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European CommissionFunding program(s)
FP7 (EU's 7th Framework Programme)
The content of the publication reflects only the author’s view. The funder is not responsible for any use that may be made of the information it contains.
Additional information about funding
AM 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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