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

Author

Listed:
  • Wiep Toorn

    (Robert Koch Institute
    Freie Universität Berlin)

  • Patrick Bohn

    (Helmholtz Centre for Infection Research)

  • Wang Liu-Wei

    (Robert Koch Institute
    Freie Universität Berlin
    International Max-Planck Research School for Biology and Computing (IMPRS-BAC))

  • Marco Olguin-Nava

    (Helmholtz Centre for Infection Research)

  • Anne-Sophie Gribling-Burrer

    (Helmholtz Centre for Infection Research
    UPR 9002)

  • Redmond P. Smyth

    (Helmholtz Centre for Infection Research
    UPR 9002
    University of Würzburg)

  • Max Kleist

    (Robert Koch Institute
    Freie Universität Berlin)

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.

Suggested Citation

  • Wiep Toorn & Patrick Bohn & Wang Liu-Wei & Marco Olguin-Nava & Anne-Sophie Gribling-Burrer & Redmond P. Smyth & Max Kleist, 2025. "Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-59102-9
    DOI: 10.1038/s41467-025-59102-9
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    References listed on IDEAS

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    1. Jiaxu Wang & Lin Yang & Anthony Cheng & Cheng-Yong Tham & Wenting Tan & Jefferson Darmawan & Paola Florez Sessions & Yue Wan, 2024. "Direct RNA sequencing coupled with adaptive sampling enriches RNAs of interest in the transcriptome," Nature Communications, Nature, vol. 15(1), pages 1-10, December.
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