Spatial isoform sequencing at single-cell resolution reveals cell-type-specific spatial isoform variability in multiple brain cell types.

Spatial long-read technologies are increasingly common but usually lack single-cell resolution. This leaves unanswered whether spatially variable isoforms reflect variability within one cell type or differences in region-specific cell-type composition. Here, we developed Spl-ISO-Seq2 (500-nm resolution) and accompanying software, Spl-IsoQuant-2 and Spl-IsoFind, enabling long-read sequencing of >450 million barcodes versus 80,000 previously. Applying this to the adult mouse brain, we compared differential isoform abundance between known regions and spatial isoform patterns independent of predefined regions. Both identified overlapping hits, for example, Rps24 in oligodendrocytes. For known Snap25 spatial isoform variation, we show that it occurs in excitatory neurons. The region-agnostic approach also uncovered patterns missed by region-based comparisons, for example, for Ighm. Notably, many spatial isoform signals are not driven by cell-type composition alone. Finally, our software is applicable to many spatial and single-cell protocols, demonstrating reproducibility between platforms (for example, Visium HD/Stereo-seq). Overall, our experimental/analytical methods enable a submicron-resolution-isoform view and open avenues for spatial isoform disease research.