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Related Concept Videos

RNA-seq03:21

RNA-seq

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 microarray-based...
Ribosome Profiling02:24

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...

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Related Experiment Video

Updated: Jul 17, 2026

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs
08:49

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

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Distinct 5' and 3' coverage biases shape transcriptome interpretation in Nanopore direct RNA versus PCR-cDNA

Rebecca E Lane1,2, Eleanor Calcutt1, Anandagopal Srinivasan1

  • 1Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Research Unit (BRU), National Institute of Health Research Oxford Biomedical, University of Oxford, Oxford, UK.

BMC Genomics
|July 15, 2026
PubMed
Summary

Choosing between PCR-cDNA and direct RNA sequencing protocols is critical for accurate long-read transcriptomics. Protocol biases significantly impact gene expression and isoform analysis, necessitating careful method selection for reliable biological insights.

Keywords:
Direct RNA-seqLong-readOxford NanoporeRNA-seq

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Last Updated: Jul 17, 2026

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs
08:49

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Published on: September 16, 2019

Sequencing of mRNA from Whole Blood using Nanopore Sequencing
11:26

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Published on: June 3, 2019

AQRNA-seq for Quantifying Small RNAs
05:12

AQRNA-seq for Quantifying Small RNAs

Published on: February 2, 2024

Area of Science:

  • Molecular Biology
  • Genomics
  • Transcriptomics

Background:

  • Long-read RNA sequencing offers isoform-resolved transcriptomics.
  • Library preparation methods introduce systematic biases.
  • These biases can influence biological interpretations.

Purpose of the Study:

  • To benchmark Oxford Nanopore's PCR-cDNA and direct RNA protocols.
  • To evaluate protocol-specific biases in long-read RNA sequencing.
  • To assess the impact of these biases on transcriptomic analysis.

Main Methods:

  • Utilized SKMM2 myeloma cells stimulated with interleukin-6 (IL-6).
  • Incorporated ERCC synthetic spike-ins for control.
  • Compared Oxford Nanopore's PCR-cDNA and direct RNA library preparation methods.

Main Results:

  • Direct RNA yielded longer, higher-quality reads but had 5' coverage loss.
  • PCR-cDNA produced shorter fragments with 3' underrepresentation, detecting more low-abundance transcripts.
  • Protocol biases led to limited overlap in differential gene expression and protocol-specific isoform usage.

Conclusions:

  • Both methods offer accurate gene-level quantification.
  • Transcript-level conclusions are highly dependent on the chosen protocol.
  • Careful protocol selection is essential for robust long-read transcriptomics.