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 6 min 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.
