Single-cell long-read transcriptomics: from technologies to biological insights
Abstract Single-cell long-read transcriptomics (scLR-seq) extends single-cell analysis beyond gene abundance by resolving full-length transcript structures in individual cells. It can directly interrogate isoform usage, alternative splicing, and transcription start and end site selection, thereby revealing regulatory variation that is often obscured by short-read measurements. In this review, we examine the experimental and computational foundations of scLR-seq, including platform selection, library design, cell barcode and unique molecular identifier recovery, transcript discovery, and isoform quantification. We discuss how these choices influence the reliability of downstream biological interpretation, and summarize emerging insights into isoform usage, alternative splicing, transcription start and end site selection, allele-specific expression, fusion transcripts, transposable element-derived transcripts, and RNA modifications. Finally, we highlight applications of scLR-seq in diverse biological systems, such as the immune system, neural development, and tumor microenvironments, and consider future opportunities and challenges in integrating multi-omics data to decode cellular programs and disease evolution.