WarpDemuX-custom - Multiplexed sequence-specific direct RNA sequencing of ribosomal RNA and beyond
Nanopore direct RNA sequencing has emerged as a powerful technology for studying RNA modifications at single- molecule resolution. Although the method was originally developed for polyadenylated RNAs, community-driven protocol adaptations have expanded its applicability to non-polyadenylated RNA species, including tRNAs and rRNAs.The lack of open (de)multiplexing solutions limits however the feasibility of large-scale sequencing studies and hampers the broader adoption of this technology. Multiplexing 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 associated with nanopore-based targeted sequencing in the absence of a poly(A) tail as is the case for rRNAs. 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 WarpDemuX toolbox to develop a (de)multiplexing strategy for sequence-specific direct RNA sequencing, WarpDemuX-custom. We validate our approach using human cell lines carrying gene knockouts for different C/D box snoRNAs, required for position-specific 2-O ribose methylations on 28S rRNA. Beyond rRNAs, it is also applicable to other non-polyadenylated RNAs, such as lncRNAs and viral RNAs.