Biology subjects

van der Toorn, W.

Publications and source records attributed to van der Toorn, W..

6 recordsLinked to original sources

bioRxiv2026

WarpDemuX-custom - Multiplexed sequence-specific direct RNA sequencing

Nanopore direct RNA sequencing has emerged as a powerful technology for studying RNA and modifications at single-molecule resolution. Although originally developed for polyadenylated RNAs, protocol adaptations have extended its application to non-polyadenylated RNA species, including tRNAs and rRNAs. However, the lack of open and flexible (de)multiplexing solutions remains a limitation for large-scale sequencing studies. Existing community-driven approaches enable multiplexing of polyadenylated RNAs and, more recently, tRNAs, but do not fully address the challenges of targeted sequence-specific Nanopore sequencing of non-polyadenylated RNAs. Here, we present an improved sequence-specific direct RNA sequencing protocol for ribosomal RNAs, achieving enhanced capture and longer read lengths as demonstrated for human 28S rRNA. We complement this protocol with an expanded WarpDemuX software toolbox for flexible sequence-specific (de)multiplexing. We validate the approach using human cell lines carrying knockouts of different C/D box snoRNAs involved in site-specific 2'-O-ribose methylation of 28S rRNA. Our results demonstrate efficient multiplexed detection of sequence-specific RNA molecules and associated modification changes. While established here using rRNA, we validate our approach on another panel of non-polyadenylated RNA species, providing an open and scalable framework for targeted direct RNA sequencing and RNA modification analysis

bioinformatics↗

bioRxiv2025

Native epitranscriptome sequencing reveals Hepatitis B virus RNA stability protected by heavy m6A modification

Although the RNA of Hepatitis B virus (HBV) is known to be regulated by the epitranscriptomic modification N6-methyladenosine (m6A), it remains controversial whether if m6A is beneficial or detrimental for viral replication. Here, we employ Nanopore direct RNA sequencing (DRS) to analyze the HBV m6A epitranscriptome on a single molecule level and elucidate how m6A regulates the HBV replication cycle. We found more than 60% of all HBV RNA in cells are m6A methylated, [~]3x more methylated than host mRNAs. Previously unreported m6A sites were found in pol gene, which when mutated reduced viral production. A sizable proportion of HBV RNAs carry poly(A) tails at least twice as long (>120nt) as cellular mRNAs, with the shorter-tailed population more pronounced among m6A-free transcripts. In contrast, encapsidated RNAs have shorter poly(A) tails and less m6A. Concordantly, pharmacological reduction of viral m6A using the m6A methyltransferase inhibitor STM2457, shortens viral poly(A) tails and reduces viral RNA stability. Our findings thus suggest that the abundance of m6A on viral RNAs protects the surprisingly long viral poly(A) tails and thus enhances RNA stability. Overall, m6A strongly enhances HBV viral replication, in support of RNA modification machinery as a promising class of antiviral targets.

microbiology↗

bioRxiv2025

WarpDemuX-tRNA: barcode multiplexing for nanopore tRNA sequencing

Transfer RNA (tRNA) plays an essential role in protein translation and tRNA modifications are important to their function. Recently, nanopore direct RNA sequencing (dRNA-seq) has shown promising results in the detection of complex tRNA modifications. However, its wider adoption in the tRNA field has been limited by a lack of (de)multiplexing solutions. Here, we present WarpDemuX-tRNA: an extension to the WarpDemuX method specifically optimized for multiplexed nanopore tRNA sequencing. Using consensus-based signal analysis using (soft) Dynamic Time Warping and barycenter averaging, our approach improves barcode feature generation and achieves more robust barcode identification. WarpDemuX-tRNA outperforms the original method and achieves 99% precision and 95% recovery for four barcodes, while reducing computational complexity and runtime to six minutes per one million reads. WarpDemuX-tRNA is an open-source and free-to-use solution to high-throughput nanopore tRNA sequencing, facilitating more accessible, cost-effective and high-throughput studies of tRNA modifications and their regulatory mechanisms.

bioinformatics↗

bioRxiv2025

MoDorado: Enhanced detection of tRNA modifications in nanopore sequencing by off-label use of modification callers

Rapid and accurate identification of tRNA modifications is crucial for understanding their role in protein translation and disease. However, their detection on tRNAs is challenging due to their high modification density. Recently, modification calling models for nanopore direct RNA sequencing became available for pseudouridine ({Psi}), m6A, inosine and m5C, as part of the Dorado basecaller. Applying the {Psi} model to tRNAs, we have mapped both known and novel {Psi} sites in Schizosaccharomyces pombe and assigned the responsible pseudouridine synthetases. Furthermore, we have developed MoDorado, an algorithm to detect modifications beyond those used in model training ("off-label use") by measuring prediction differences of pre-trained machine learning models. By leveraging the {Psi}/m6A/inosine/m5C models, MoDorado detected seven additional modifications (ncm5U, mcm5U, mcm5s2U, m7G, queuosine, m1A, and i6A), thus generating a tRNA modification map of S. pombe. This work demonstrates the potential of pre-trained models in determining the intricate landscape of tRNA modifications.

molecular biology↗

bioRxiv2024

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

Nanopore direct RNA sequencing (dRNA-seq) enables unique insights into (epi-)transcriptomics. However, applications are currently limited by the lack of accurate and cost-effective sample multiplexing. We introduce WarpDemuX, an ultra-fast and highly accurate adapter-barcoding and demultiplexing approach. WarpDemuX enhances speed and accuracy by fast processing of the raw nanopore signal, use of a lightweight machine-learning algorithm and design of optimized barcode sets. We demonstrate its utility by performing a rapid phenotypic profiling of different SARS-CoV-2 viruses, crucial for pandemic prevention and response, through multiplexed sequencing of longitudinal samples on a single flowcell. This identifies 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 is a broadly applicable, high-performance, and economical multiplexing solution for nanopore dRNA-seq, facilitating advanced (epi-)transcriptomic research.

bioinformatics↗

bioRxiv2023

Sequencing accuracy and systematic errors in nanopore direct RNA sequencing

Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been exploring and expanding the potential of dRNA-seq, the sequencing accuracy and error patterns remain understudied. We present the first comprehensive evaluation of accuracy and systematic errors in dRNA-seq data from diverse species, as well as synthetic RNA. Deletions significantly outnumbered mismatches/insertions, while the median read accuracy exhibited species-level variation. In addition to homopolymer errors, we observed systematic biases across nucleotides and heteropolymeric motifs in all species. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines/adenines. Moreover, the systematic errors were strongly dependent on local sequence contexts. By examining raw signal data, we identified underlying signal-level features potentially associated with the error patterns. While read quality scores approximated error rates at base and read levels, failure to detect DNA adapters may lead to data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (base-calling) artefacts. Lastly, we discuss the implications of such error patterns for downstream applications of dRNA-seq data.

bioinformatics↗