Spatial isoform sequencing at sub-micrometer single-cell resolution reveals novel patterns of spatial isoform variability in brain cell types.

Spatial long-read technologies are becoming more common but lack nanometer- and therefore often single-cell resolution. This leaves the question unanswered whether spatially variable isoforms represent spatial variability within one cell type or differences in cell-type abundance between different regions. Here, we develop Spl-ISO-Seq2 with 220nm spot size and 500nm resolution, and the accompanying software packages Spl-IsoQuant-2 and Spl-IsoFind and apply it to the adult mouse brain. We compare spatial variability within a fixed cell type by examining (a) differential isoform abundance between known brain regions and (b) spatial isoform patterns that do not align with predefined regions. The former reveals larger numbers of spatial isoform differences, e.g. Rps24 in oligodendrocytes. For the previously appreciated gene with spatially-variable isoforms Snap25, we can now show that this variability exists in excitatory neurons. However, the latter approach reveals patterns that the former cannot conceptually model, e.g., Tnnc1 in excitatory neurons. Taken together, our experimental and analytical methods enrich spatial transcriptomics with a so-far elusive isoform view of spatial variation for individual cell types.