Nanopore direct RNA sequencing (dRNA-seq) enables unique insights into RNA biology. However, applications are currently limited by the lack of accurate and cost-effective sample multiplexing. Here we introduce WarpDemuX, an ultra-fast and highly accurate adapter-barcoding and demultiplexing approach for dRNA-seq with SQK-RNA002 and SQK-RNA004 chemistries. WarpDemuX enhances speed and accuracy by fast processing of the raw nanopore signal, use of a light-weight machine-learning algorithm and design of optimized barcode sets. We demonstrate its utility by performing rapid phenotypic profiling of different SARS-CoV-2 viruses through multiplexed sequencing of longitudinal samples on a single flowcell, identifying systematic differences in transcript abundance and poly(A) tail lengths during infection. Additionally, integrating WarpDemuX into sequencing control software enables real-time enrichment of target molecules through barcode-specific adaptive sampling, which we demonstrate by enriching low abundance viral RNA. In summary, WarpDemuX represents a broadly applicable, high-performance, economical multiplexing solution for dRNA-seq, facilitating advanced (epi-) transcriptomic research.
