Direct RNA sequencing enables improved transcriptome assessment and tracking of RNA modifications for medical applications
Direct RNA sequencing (DRS) is a Nanopore-based technique for analyzing RNA in its native form. This technique promises breakthroughs in diagnostics and biomarker development. Coupled to RNA002 sequencing chemistry, its clinical implementation has been challenging due to low throughput, low accuracy, and lack of large-scale RNA-modification models. In this study, we evaluate the improvements achieved by pairing the latest RNA004 chemistry with novel modified-base-calling models for pseudouridine and N6-methyladenosine using diverse RNA samples from cell lines, synthetic oligos, and human blood. Finally, we present the first clinical application of DRS by confirming the loss of RNA methylation in a patient carrying truncating mutations in the methyltransferase METTL5. Conclusively, the combined use of RNA004 chemistry with the base-calling models significantly improved the throughput, accuracy, and site-specific detection of modifications. From this perspective, we offer an outlook on the potential suitability of DRS for use in routine diagnostics, as well as the first comprehensive benchmark of human peripheral blood. Furthermore, we demonstrate on the basis of a stop-codon readthrough enhancing agent potential roadblocks for routine quality assessments of RNA therapeutics.
Authors
- Mark Helm (ORCID: https://orcid.org/0000-0002-0154-0928)
- Susanne Gerber (ORCID: https://orcid.org/0000-0001-9513-0729)
- Kristi Jensen (ORCID: https://orcid.org/0000-0002-9414-8043)
- Vincent Dietrich (ORCID: https://orcid.org/0000-0003-0568-9727)
- Stefan Mündnich
- Stephan Weißbach (ORCID: https://orcid.org/0009-0005-4347-952X)
- Susann Schweiger (ORCID: https://orcid.org/0000-0002-7816-8984)
- Florian Kraft (ORCID: https://orcid.org/0000-0002-5324-9155)
- Verena Haug (ORCID: https://orcid.org/0000-0003-4071-8482)
- Stanislav Sys (ORCID: https://orcid.org/0000-0001-5402-1542)
- Edward A. Lemke (ORCID: https://orcid.org/0000-0002-0634-0503)
- Jonas P. Koch (ORCID: https://orcid.org/0000-0003-1833-9064)
- Matthias Linke (ORCID: https://orcid.org/0000-0002-7494-9812)
- Charlotte Hewel (ORCID: https://orcid.org/0000-0003-0967-0054)
- Felix Hofmann (ORCID: https://orcid.org/0000-0002-3891-6239)
- Laura Holthöfer (ORCID: https://orcid.org/0009-0003-2441-5251)
- Anna Wierczeiko (ORCID: https://orcid.org/0000-0003-0684-926X)
- Norbert W. Paul (ORCID: https://orcid.org/0000-0002-8288-6727)
- Frank Lyko (ORCID: https://orcid.org/0000-0002-4873-5431)
- Stefan Diederich (ORCID: https://orcid.org/0000-0003-3971-6498)
- Alexandra Russo (ORCID: https://orcid.org/0009-0000-1141-7977)
- Lukas Schartel
- Tamer Butto (ORCID: https://orcid.org/0000-0001-8028-0038)
- Johannes Friedrich (ORCID: https://orcid.org/0000-0002-1321-5866)
- Johannes Miedema
- Lioba Lehmann
Institutions
- Max Planck Society (DE)
- German Cancer Research Center (DE)
- Johannes Gutenberg University Mainz (DE)
- Heidelberg University (DE)
- University Hospital Heidelberg (DE)
- University Medical Center of the Johannes Gutenberg University Mainz (DE)
- Quantitative BioSciences (US)
- Universitätsklinikum Aachen (DE)
- DKFZ-ZMBH Alliance (DE)
- University Medical Centre Mannheim (DE)
- Institute of Molecular Biology (DE)
- Heidelberg University (US)
- RWTH Aachen University (DE)
Publication Details
- Journal
- Nucleic Acids Research
- Published
- 2025-11-05
- DOI
- https://doi.org/10.1093/nar/gkaf1314
- Citations
- 4
- Primary Topic
- RNA modifications and cancer
- Type
- article
- Field-Weighted Citation Impact
- 3.22