Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing

Wiep Toorn, Patrick Bohn, Wang Liu-Wei, Marco Olguin-Nava, Anne-Sophie Gribling-Burrer, Redmond P. Smyth () and Max Kleist ()
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Wiep Toorn: Robert Koch Institute
Patrick Bohn: Helmholtz Centre for Infection Research
Wang Liu-Wei: Robert Koch Institute
Marco Olguin-Nava: Helmholtz Centre for Infection Research
Anne-Sophie Gribling-Burrer: Helmholtz Centre for Infection Research
Redmond P. Smyth: Helmholtz Centre for Infection Research
Max Kleist: Robert Koch Institute

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-59102-9

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