Directional high-throughput sequencing of RNAs without gene-specific primers

Content Provider	Directory of Open Access Journals (DOAJ)
Author	Anita Mäki Marja Tiirola
Abstract	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 workflow 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 profile 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.
Related Links	https://www.future-science.com/doi/10.2144/btn-2018-0082
ISSN	07366205
e-ISSN	19409818
DOI	10.2144/btn-2018-0082
Journal	BioTechniques
Issue Number	4
Volume Number	65
Language	English
Publisher	Future Science Ltd
Publisher Date	2018-01-01
Publisher Place	United Kingdom
Access Restriction	Open
Subject Keyword	Biology (general) Algae Ligation Next-generation Sequencing Phytoplankton Primer Bias Random Priming
Content Type	Text
Resource Type	Article
Subject	Biotechnology