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

Biotechniques. 2018 Oct;65(4):219-223. doi: 10.2144/btn-2018-0082.


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.

Keywords: algae; ligation; next-generation sequencing; phytoplankton; primer bias; random priming; ribosomal RNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Primers / genetics
  • DNA, Complementary / genetics
  • Gene Library*
  • High-Throughput Nucleotide Sequencing / methods*
  • Phytoplankton / genetics
  • RNA, Ribosomal / genetics*
  • Workflow


  • DNA Primers
  • DNA, Complementary
  • RNA, Ribosomal