Long-read transcriptomics - opportunities and challenges

Long-read RNA sequencing resolves full-length transcript structures, complex splicing, alternative boundaries, transcriptional overlaps, and multigenic isoforms without short-read reconstruction. Current approaches include PacBio HiFi and Kinnex cDNA sequencing, Oxford Nanopore cDNA sequencing, and direct RNA sequencing of native molecules. We critically review platform-specific trade-offs, analytical workflows, sequencing-depth requirements, and biomedical applications, and provide practical guidance for robust transcript discovery, quantification, rare-disease interpretation, viral transcriptomics, and single-cell or spatial isoform analysis.