bioRxiv : the preprint server for biologyJul 14, 2025

Assessing improvements in nanopore direct RNA sequencing for detecting RNA modifications

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Mar 7, 2026

Abstract

Dorado's pseudouridine model achieved 96-98% accuracy and F1-score in detecting RNA modifications.

Nanopore technology enables the sequencing of intact RNA molecules, allowing for the reading of native modifications.
The updated RNA sequencing chemistry (RNA004) includes an improved basecaller (Dorado) that enhances sequencing accuracy.
Dorado demonstrated significant differences in modification calling accuracy compared to the previous RNA002 chemistry.
Both pseudouridine and N6-methyladenosine models from Dorado showed high accuracy and F1-scores ranging from 94-99%.
The technology could potentially identify multiple RNA modifications on individual mRNA strands.
Validation of modification calls through additional methods is necessary for increased confidence in results.

Simplified

Nanopore technology can directly sequence intact RNA molecules, offering a unique capability to read native modifications. Oxford Nanopore Technologies recently updated its direct RNA sequencing technology from RNA002 to RNA004 chemistry. This update included an improved basecaller (Dorado) for increased sequencing accuracy, and ionic current models fordetection of four RNA modifications. Using a single RNA extraction from GM12878 B-lymphocyte cell line, we compared RNA002 and RNA004 sequencing chemistries and evaluated Dorado modification calling accuracy. We computed U-to-C mismatches, previously used to identify putative pseudouridine sites, and ran m6anet for identifying putative N6-methyladenosine sites. Dorado results for each respective modification showed both global and site-specific differences when compared to RNA002 results. We used DRS data fromtranscription of GM12878 genomic DNA as well as synthetic oligonucleotides to evaluate Dorado modification calling performance. Dorado's pseudouridine model achieved 96-98% for both accuracy and F1-score. Similarly, Dorado's N6-methyladenosine model achieved 94-98% accuracy, 96-99% F1-score. Our results demonstrate that Nanopore Direct RNA sequencing could simultaneously detect pseudouridine, N6-methyladenosine, 5-methylcytosine, and inosine modifications on individual mRNA strands. It is critical to validate these calls using orthogonal methods (e.g., Liquid Chromatography with Tandem Mass Spectrometry) for increased confidence. de novo in vitro

Full Text

Full text is available at the source.

View Full Text ↗

Funding

Competing interests

Competing interest statement: M.J. is a consultant to ONT and has received reimbursement for travel, accommodation, and conference fees to speak at events. Other authors declare no competing interests.

PubMed

Assessing improvements in nanopore direct RNA sequencing for detecting RNA modifications

Abstract

Full Text

Funding

Competing interests

You found one interesting study. We’ll send the next 7.

What Lands in Your Inbox Each Week:

Recent issues from the mrna technology brief

Abstract

Full Text

Funding

Competing interests

Related papers

You found one interesting study. We’ll send the next 7.

What Lands in Your Inbox Each Week:

Recent issues from the mrna technology brief