A long-read-compatible spatial barcoding system: enables isoform-resolved spatial heterogeneity at near-single-cell scale.

We developed a spatial barcoding system compatible with long-read sequencing, combining high-density capture arrays with a three-segment combinatorial barcoding strategy. This framework enables large-scale spatial sampling while maintaining low barcode collision.

In the in situ cDNA synthesis stage, spatial coordinates are assigned, allowing the same library to be used for robust Illumina quantification and full-length Nanopore sequencing. We generated near-single-cell resolution spatial maps in tomato-pepper grafts and mouse embryos, demonstrating high consistency between NGS and TGS data. This platform extends spatial transcriptomics from gene-level expression to isoform-resolved in situ biology.