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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年4月21日

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  • Wiep van der Toorn,
  • Patrick Bohn,
  • Wang Liu-Wei,
  • Marco Olguin-Nava,
  • Anne-Sophie Gribling-Burrer,
  • Redmond P. Smyth,
  • Max von Kleist

摘要

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.