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Related Concept Videos

RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
9.2K

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Related Experiment Video

Updated: Apr 27, 2026

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs
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Evaluating bias-reducing protocols for RNA sequencing library preparation.

Thomas J Jackson1, Ruth V Spriggs, Nicholas J Burgoyne

  • 1Medical Research Council Toxicology Unit, Lancaster Rd, Leicester LE1 9HN, UK. mzyatjj@nottingham.ac.uk.

BMC Genomics
|July 9, 2014
PubMed
Summary
This summary is machine-generated.

A new CircLigase protocol for RNA sequencing library preparation reduces bias in Ion Torrent PGM data. This single adaptor approach improves read abundance accuracy but does not fully eliminate sequence over-representation.

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Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing
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Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Next-generation sequencing (NGS) can produce biased read abundance estimates, potentially affecting data interpretation.
  • The RNA sequencing (RNA-Seq) library ligation step is a known source of bias.
  • Investigating alternative library construction protocols is crucial for improving NGS accuracy.

Purpose of the Study:

  • To compare the standard duplex adaptor protocol with a single adaptor CircLigase (CircLig) protocol for Ion Torrent PGM sequencing.
  • To assess if using a thermostable ligase can mitigate bias related to RNA secondary structure.

Main Methods:

  • A small RNA pool of known composition was used to generate sequencing libraries.
  • Three library preparation protocols were tested, including the standard Life Technologies duplex adaptor and the CircLig protocol.
  • Sequencing was performed on the Ion Torrent PGM platform.

Main Results:

  • The CircLig protocol demonstrated reduced over-representation of specific sequences compared to the standard protocol.
  • Sequences showing over-representation were predicted to have secondary structures and co-fold with adaptors.
  • A thermostable ligase (Methanobacterium thermoautotrophicum RNA ligase K97A - Mth K97A) did not effectively reduce bias.

Conclusions:

  • The single adaptor CircLigase approach significantly reduces, but does not eliminate, bias in Ion Torrent sequencing data.
  • Ligases functioning at higher temperatures to disrupt secondary structures may offer value, though Mth K97A was ineffective in this study.