Systematic benchmarking of commercial workflows for isoform-resolved single-nucleus transcriptomics

Abstract

Short-read sequencing-based single-cell transcriptomics represents the current gold standard for studying cellular transcriptomes but remains limited in its ability to resolve full-length transcript isoforms and splicing patterns. Long-read single-cell and single-nucleus RNA sequencing (LR sc/snRNA-seq) enables the transcriptome-wide characterization of full-length isoforms at cellular resolution, yet the relative performance of commercially available workflows remains insufficiently explored. Here, using nuclei extracted from a standardized multi-species benchmark sample and Oxford Nanopore Technologies long-read sequencing, we systematically benchmarked four LR snRNA-seq strategies: 10x Genomics 3', 10x Genomics 5', ArgenTag, and Parse Biosciences. Comparing transcriptome features qualitatively and quantitatively, as well as the concordance with matched short-read data and the ability to resolve cellular heterogeneity, we identified substantial method-specific differences in read length and yield, transcript coverage, isoform detection, and recovery of sample-specific biological information, with the 10x Genomics 3' and 5' assays emerging as the most balanced approaches for comprehensive isoform-resolved single-nucleus transcriptomics. Altogether, our study provides a systematic assessment of four commercially available workflows for performing LR snRNA-seq and highlights key methodological trade-offs related to distinct library preparation strategies, thus providing practical guidance for future isoform-resolved transcriptome studies at the single-nucleus level.