Direct RNA sequencing enables improved transcriptome assessment and tracking of RNA modifications for medical applications

Nucleic Acids Research, vol. 53

Abstract

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 21

1. Charlotte Hewel

   Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

   Affiliation as printed

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz , Mainz 55131 ,

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55131, Germany

2. Anna Wierczeiko

   Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

   Affiliation as printed

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz , Mainz 55131 ,

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55131, Germany

3. Johannes Miedema

   Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

   Affiliation as printed

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz , Mainz 55131 ,

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55131, Germany

4. Johannes H. Friedrich

   Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

   Affiliation as printed

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz , Mainz 55131 ,

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55131, Germany

5. Stephan Weißbach

   Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

   Affiliation as printed

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz , Mainz 55131 ,

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55131, Germany

6. Laura Holthöfer

   Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

   Affiliation as printed

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz , Mainz 55131 ,

   Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55131, Germany

7. Verena Haug

   Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

   Affiliation as printed

   Department for Pediatrics, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz ,

   Department for Pediatrics, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany

8. Stefan Mündnich

   Johannes Gutenberg University Mainz

   Affiliation as printed

   Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz , Mainz 55128 ,

   Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Mainz 55128, Germany

9. Lukas Schartel

   Max Planck Society · Johannes Gutenberg University Mainz

   Affiliation as printed

   Biocenter, Johannes Gutenberg University Mainz , Hanns-Dieter-Hüsch-Weg 17, Mainz 55128 ,

   International Max Planck Research School on Cellular Biophysics , Hanns-Dieter-Hüsch-Weg 17, Mainz 55128 ,

10. Stanislav Jur`Evic Sys

    Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

    Affiliation as printed

    Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz , Mainz 55131 ,

    Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55131, Germany

11. Tamer Butto

    Johannes Gutenberg University Mainz

    Affiliation as printed

    Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz , Mainz 55128 ,

    Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Mainz 55128, Germany

12. Norbert W. Paul

    Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

    Affiliation as printed

    Institute for the History, Philosophy, and Ethics of Medicine, Johannes Gutenberg University Medical Center Mainz , Mainz 55131 ,

    Institute for the History, Philosophy, and Ethics of Medicine, Johannes Gutenberg University Medical Center Mainz, Mainz 55131, Germany

13. Jonas P. Koch

    German Cancer Research Center · Heidelberg University · DKFZ-ZMBH Alliance

    Affiliation as printed

    Division of Epigenetics, DKFZ–ZMBH Alliance, German Cancer Research Center , Heidelberg 69120 ,

    Faculty of Biosciences, Heidelberg University , Heidelberg, 69120 ,

14. Frank Lyko

    German Cancer Research Center · DKFZ-ZMBH Alliance · University Medical Centre Mannheim

    Affiliation as printed

    DKFZ Hector Cancer Institute at the University Medical Center Mannheim , Heidelberg 69120 ,

    Division of Epigenetics, DKFZ–ZMBH Alliance, German Cancer Research Center , Heidelberg 69120 ,

    DKFZ Hector Cancer Institute at the University Medical Center Mannheim, Heidelberg 69120, Germany

15. Florian Kraft Aachen Institut für Humangenetik und Genommedizin

    Universitätsklinikum Aachen · RWTH Aachen University

    Affiliation as printed

    Institut für Humangenetik und Genommedizin, Uniklinik RWTH Aachen , Aachen ,

16. Alexandra Russo

    Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

    Affiliation as printed

    Department for Pediatrics, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz ,

    Department for Pediatrics, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany

17. Susann Schweiger

    Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

    Affiliation as printed

    Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz , Mainz 55131 ,

    Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55131, Germany

18. Edward A. Lemke

    Johannes Gutenberg University Mainz · Institute of Molecular Biology

    Affiliation as printed

    Biocenter, Johannes Gutenberg University Mainz , Hanns-Dieter-Hüsch-Weg 17, Mainz 55128 ,

    Institute for Quantitative and Computer Biosciences (IQCB) , Mainz 55128 ,

    Institute of Molecular Biology (IMB), Ackermannweg 4 , Mainz 55128 ,

    Institute for Quantitative and Computer Biosciences (IQCB), Mainz 55128, Germany

19. Mark Helm

    Johannes Gutenberg University Mainz

    Affiliation as printed

    Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz , Mainz 55128 ,

    Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Mainz 55128, Germany

20. Matthias Linke

    Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

    Affiliation as printed

    Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz , Mainz 55131 ,

    Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55131, Germany

21. Susanne Gerber corresponding

    Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

    Affiliation as printed

    Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz , Mainz 55131 ,

    Institute for Quantitative and Computer Biosciences (IQCB) , Mainz 55128 ,

    Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55131, Germany

    Institute for Quantitative and Computer Biosciences (IQCB), Mainz 55128, Germany