WarpDemuX-custom - Multiplexed sequence-specific direct RNA sequencing
- 1Universitat Heidelberg
Abstract
Nanopore direct RNA sequencing has emerged as a powerful technology for studying RNA and modifications at single-molecule resolution. Although originally developed for polyadenylated RNAs, protocol adaptations have extended its application to non-polyadenylated RNA species, including tRNAs and rRNAs. However, the lack of open and flexible (de)multiplexing solutions remains a limitation for large-scale sequencing studies. Existing community-driven approaches enable multiplexing of polyadenylated RNAs and, more recently, tRNAs, but do not fully address the challenges of targeted sequence-specific Nanopore sequencing of non-polyadenylated RNAs. Here, we present an improved sequence-specific direct RNA sequencing protocol for ribosomal RNAs, achieving enhanced capture and longer read lengths as demonstrated for human 28S rRNA. We complement this protocol with an expanded WarpDemuX software toolbox for flexible sequence-specific (de)multiplexing. We validate the approach using human cell lines carrying knockouts of different C/D box snoRNAs involved in site-specific 2'-O-ribose methylation of 28S rRNA. Our results demonstrate efficient multiplexed detection of sequence-specific RNA molecules and associated modification changes. While established here using rRNA, we validate our approach on another panel of non-polyadenylated RNA species, providing an open and scalable framework for targeted direct RNA sequencing and RNA modification analysis
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BibTeX
@misc{vries2026warpdemuxcustom,
title = {{WarpDemuX-custom - Multiplexed sequence-specific direct RNA sequencing}},
author = {I. Naarmann-de Vries and W. van der Toorn and S. Mishra and M. F. Blank and D. Heid and C. Mueller-Tidow and M. von Kleist and C. Dieterich},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.09.757944},
url = {https://www.biorxiv.org/content/10.64898/2026.10.09.757944v1},
note = {CC BY-ND},
}
RIS
TY - UNPB
TI - WarpDemuX-custom - Multiplexed sequence-specific direct RNA sequencing
AU - I. Naarmann-de Vries
AU - W. van der Toorn
AU - S. Mishra
AU - M. F. Blank
AU - D. Heid
AU - C. Mueller-Tidow
AU - M. von Kleist
AU - C. Dieterich
PY - 2026
DA - 2026/10/09
PB - bioRxiv
DO - 10.64898/2026.10.09.757944
UR - https://www.biorxiv.org/content/10.64898/2026.10.09.757944v1
AB - Nanopore direct RNA sequencing has emerged as a powerful technology for studying RNA and modifications at single-molecule resolution. Although originally developed for polyadenylated RNAs, protocol adaptations have extended its application to non-polyadenylated RNA species, including tRNAs and rRNAs. However, the lack of open and flexible (de)multiplexing solutions remains a limitation for large-scale sequencing studies. Existing community-driven approaches enable multiplexing of polyadenylated RNAs and, more recently, tRNAs, but do not fully address the challenges of targeted sequence-specific Nanopore sequencing of non-polyadenylated RNAs. Here, we present an improved sequence-specific direct RNA sequencing protocol for ribosomal RNAs, achieving enhanced capture and longer read lengths as demonstrated for human 28S rRNA. We complement this protocol with an expanded WarpDemuX software toolbox for flexible sequence-specific (de)multiplexing. We validate the approach using human cell lines carrying knockouts of different C/D box snoRNAs involved in site-specific 2'-O-ribose methylation of 28S rRNA. Our results demonstrate efficient multiplexed detection of sequence-specific RNA molecules and associated modification changes. While established here using rRNA, we validate our approach on another panel of non-polyadenylated RNA species, providing an open and scalable framework for targeted direct RNA sequencing and RNA modification analysis
ER -