TNRSim - a simulator of truncated oxford nanopore direct RNA long reads for isoform analysis.

Introduction: Nanopore sequencing opens up new opportunities in transcriptome research due to the possibility of generating full-length isoform reads. However, currently available simulation software does not fully reproduce read truncation, a key feature of transcriptomic nanopore libraries. Methods: We developed TNRSim (Truncated Nanopore Reads Simulator), a nanopore transcriptome library simulator designed to reproduce realistic read-length distributions, sequencing errors, and quality scores. We further used simulated datasets with known isoform abundances to benchmark widely used isoform quantification tools. Results: TNRSim reproduced characteristic read-truncation patterns observed in empirical direct RNA sequencing datasets and provided improvements in read-length distribution, mismatch, and quality-score simulation compared with existing approaches. In a closed-set benchmark of isoform quantification methods, oarfish achieved the highest quantification accuracy among the tested tools. Discussion: TNRSim provides a framework for realistic simulation of transcriptomic nanopore sequencing data and enables controlled benchmarking of computational methods for isoform-level analysis.