ncRNAseq: simple modifications to RNA-seq library preparation allow recovery and analysis of mid-sized non-coding RNAs

Biotechniques. 2022 Jan;72(1):21-28. doi: 10.2144/btn-2021-0035. Epub 2021 Nov 29.


Despite their abundance, mid-sized RNAs (30-300 nt) have not been extensively studied by high-throughput sequencing, mostly due to selective loss in library preparation. The authors propose simple and inexpensive modifications to the Illumina TruSeq protocol (ncRNAseq), allowing the capture and sequencing of mid-sized non-coding RNAs without detriment to the coverage of coding mRNAs. This protocol is coupled with a two-step alignment: a pre-alignment to a curated non-coding genome, passing only the non-mapping reads to a standard genomic alignment. ncRNAseq correctly assigns the highest read-numbers to established abundant non-coding RNAs and correctly identifies cytosolic and nuclear enrichment of known non-coding RNAs in two cell lines.

Keywords: 5′ coverage; high-throughput sequencing; next-generation sequencing; non-coding RNA; size-selection.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Library
  • High-Throughput Nucleotide Sequencing* / methods
  • RNA, Untranslated*
  • RNA-Seq
  • Sequence Analysis, RNA / methods


  • RNA, Untranslated