WarpDemuX-rRNA - Multiplexed sequence-specific direct RNA sequencing of ribosomal RNA and beyond

Background: Nanopore direct RNA sequencing has emerged as a powerful technology for studying RNA modifications at single-molecule resolution. Although the method was originally developed to sequence polyadenylated RNAs, community-driven protocol adaptations have expanded its applicability to non-polyadenylated RNA species, including tRNAs and rRNAs. Despite these advances, the lack of open (de)multiplexing solutions limits the feasibility of large-scale sequencing studies and hampers the broader adoption of this technology. Multiplexing reduces sequencing costs by enabling the simultaneous analysis of multiple samples. This is accomplished by incorporating unique identifier sequences (barcodes) into each sample prior to sequencing. Community-driven approaches have enabled multiplexed sequencing of both polyadenylated RNAs and tRNAs. However, these strategies do not address the specific challenges asso- ciated with nanopore-based targeted sequencing in the the absence of a poly(A) tail as is the case for rRNAs. Results: Here, we present an improved sequence-specific Nanopore rRNA sequencing protocol with enhanced capture of the human 28S rRNA and longer read lengths. We complement this optimized protocol with an expanded WarpDe-muX toolbox to develop a (de)multiplexing strategy for sequence-specific direct RNA sequencing, WarpDemuX-rRNA. We validate our approach on a real-world application using human cell lines carrying gene knockouts for different C/D box snoRNAs. These snoRNAs are required for position-specific 2’-O ribose methylations on 28S rRNA. Conclusion: WarpDemuX-rRNA is a powerful demultiplexing tool that enables a higher throughput and more cost-effective sequencing of non-polyadenylated RNA species. Beyond rRNAs, it is also applicable to other non-polyadenylated RNAs, such as lncRNAs and viral RNAs.