Long-Read Sequencing Reveals Tumor-Specific Splicing Isoforms as Therapeutic Targets in Non–Small-Cell Lung Cancer

Despite extensive transcriptomic alterations observed in tumors, the global landscape of isoform-level alternative splicing in cancer remains largely unexplored. Leveraging long-read sequencing, we successfully identified and characterized full-length isoforms, together with tumor-specific splicing events in non-small-cell lung cancer (NSCLC). Our analysis identified 38,058 previously unannotated isoforms, which were subsequently validated using orthogonal multiomics datasets to confirm their transcriptional and translational activities. Notably, 269 splicing events were characterized as tumor specific, with 17 showing significant associations with NSCLC subtypes, while 13 were enriched across all NSCLC cases. Among these events, skipped exons in IFI27, PUF60, and ANAPC11, as well as an alternative first exon in YBEY, were absent from GENCODE annotations. These findings underscore the intricate complexity of isoforms and their clinical significance, particularly in terms of NSCLC subtype specificity and their potential as therapeutic targets. In conclusion, this study provides a valuable resource for the discovery of tumor-specific splicing targets in NSCLC, leveraging the unique advantages of long-read sequencing.