van der Toorn Wiep, Bohn Patrick, Liu-Wei Wang, Olguin-Nava Marco, Gribling-Burrer Anne-Sophie, Smyth Redmond P, von Kleist Max
Systems Medicine of Infectious Disease (P5), Robert Koch Institute, Berlin, Germany.
Department of Mathematics and Computer Science, Freie Universität Berlin, Berlin, Germany.
Nat Commun. 2025 Apr 21;16(1):3742. doi: 10.1038/s41467-025-59102-9.
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.
纳米孔直接RNA测序（dRNA-seq）能够为RNA生物学提供独特的见解。然而，目前由于缺乏准确且经济高效的样本多重分析方法，其应用受到限制。在此，我们介绍WarpDemuX，这是一种用于dRNA-seq的超快速且高度准确的接头条形码标记和多重分析方法，适用于SQK-RNA002和SQK-RNA004化学方法。WarpDemuX通过快速处理原始纳米孔信号、使用轻量级机器学习算法以及设计优化的条形码集，提高了速度和准确性。我们通过在单个流动槽上对纵向样本进行多重测序，对不同的严重急性呼吸综合征冠状病毒2（SARS-CoV-2）病毒进行快速表型分析，证明了其效用，确定了感染期间转录本丰度和多聚腺苷酸（poly(A)）尾长度的系统差异。此外，将WarpDemuX集成到测序控制软件中，能够通过条形码特异性自适应采样实现目标分子的实时富集，我们通过富集低丰度病毒RNA证明了这一点。总之，WarpDemuX代表了一种广泛适用、高性能、经济的dRNA-seq多重分析解决方案，有助于推进（表观）转录组学研究。
