Isoform-resolved spatial transcriptomics on a lab-made high-density array via a single-chip NGS-TGS workflow

Spatial transcriptomics maps gene expression within tissue sections, but dominant short-read approaches have limited ability to resolve full-length transcript structures such as alternative splicing. Long-read sequencing provides transcript-structure information, although its higher sequencing error rates can complicate spatial-barcode identification on high-density arrays. Here, we show a lab-made, high-density spatial transcriptomics system that uses a tri-part combinatorial barcode design to expand barcode capacity and facilitate spatial assignment of long reads. By splitting full-length complementary DNA generated from a single capture chip, the workflow combines high-depth short-read quantification with single-molecule long-read profiling within the same spatial coordinate system. In incompatible tomato–pepper grafts and mouse embryos, the approach identifies spatially localized unannotated isoforms and intron-retention events, and reveals regional differences in isoform usage that are not apparent from gene-level expression alone. Together, this system provides a framework for combining spatial gene-expression quantification with full-length transcript-structure analysis in the same tissue. Existing spatial transcriptomics technologies typically rely on either short-read or long-read sequencing platforms. Here, the authors develop a high-density spatial transcriptomics system that integrates short-read quantitative profiling with long-read transcript isoform analysis at the same spatial coordinates.