Rolling circle reverse transcription enables high fidelity nanopore sequencing of small RNA

PLoS One. 2022 Oct 10;17(10):e0275471. doi: 10.1371/journal.pone.0275471. eCollection 2022.

Abstract

Small RNAs (sRNAs) are an important group of non-coding RNAs that have great potential as diagnostic and prognostic biomarkers for treatment of a wide variety of diseases. The portability and affordability of nanopore sequencing technology makes it ideal for point of care and low resource settings. Currently sRNAs can't be reliably sequenced on the nanopore platform due to the short size of sRNAs and high error rate of the nanopore sequencer. Here, we developed a highly efficient nanopore-based sequencing strategy for sRNAs (SR-Cat-Seq) in which sRNAs are ligated to an adapter, circularized, and undergo rolling circle reverse transcription to generate concatemeric cDNA. After sequencing, the resulting tandem repeat sequences within the individual cDNA can be aligned to generate highly accurate consensus sequences. We compared our sequencing strategy with other sRNA sequencing methods on a short-read sequencing platform and demonstrated that SR-Cat-Seq can obtain low bias and highly accurate sRNA transcriptomes. Therefore, our method could enable nanopore sequencing for sRNA-based diagnostics and other applications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers
  • DNA, Complementary / genetics
  • High-Throughput Nucleotide Sequencing / methods
  • Nanopore Sequencing*
  • RNA, Small Untranslated* / genetics
  • Reverse Transcription

Substances

  • Biomarkers
  • DNA, Complementary
  • RNA, Small Untranslated

Grants and funding

New England Biolabs (www.neb.com) has funded this study. SM and SG are employees of New England Biolabs. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.