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Related Experiment Video

Updated: Dec 19, 2025

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing
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A low-bias and sensitive small RNA library preparation method using randomized splint ligation.

Sean Maguire1, Gregory J S Lohman1, Shengxi Guan1

  • 1New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.

Nucleic Acids Research
|June 5, 2020
PubMed
Summary

This study introduces a new small RNA sequencing method to overcome low sensitivity and bias in current techniques. The improved workflow accurately captures diverse small RNAs, including 2'-O-methyl modified RNA, for better disease research.

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Area of Science:

  • Molecular Biology
  • Genomics
  • RNA Biology

Background:

  • Small RNAs regulate gene expression in development and disease.
  • Next-generation sequencing (NGS) for small RNA studies faces sensitivity and bias limitations.
  • Ligation bias during library preparation, especially with 2 -O-methyl (2 OMe) modifications, hinders accurate small RNA profiling.

Purpose of the Study:

  • To develop a novel library preparation method for unbiased and sensitive small RNA sequencing.
  • To improve the accurate capture of diverse small RNA species, including modified RNAs.
  • To enhance the detection of differential microRNA (miRNA) expression in disease tissues.

Main Methods:

  • Development of a new library preparation workflow utilizing randomized splint ligation with a cleavable adapter.
  • Application of the workflow to small RNA sequencing.
  • Comparison of the new method against existing techniques for sensitivity and bias reduction.

Main Results:

  • The novel workflow significantly reduces ligation bias in small RNA sequencing.
  • Increased sensitivity was observed for various small RNAs, including microRNA (miRNA), tRNA fragments, and 2 OMe-modified RNAs (e.g., Piwi-interacting RNA, plant miRNA).
  • The method detected more differentially expressed miRNAs between tumorous and matched normal tissues.

Conclusions:

  • The randomized splint ligation workflow provides highly accurate small RNA sequencing.
  • This method enables detailed studies of 2 OMe modified RNAs.
  • The improved accuracy facilitates more robust discovery of disease-related small RNA biomarkers.