Extended Data Fig. 2: Comparison of spatial transcript diversity detection in adult mouse brain across ST platforms.

(a) Spatially variable exon overlaps between ONT and short-read (SR) detected variable events. Exons were decomposed from isoforms of 150 ONT-SVP genes (Fig. 3) and TREND events from 815 SR-SVP genes (Fig. 4). (b) Spatially variably processed (SVP) gene overlap between ONT and SR mouse CBS samples. Events were identified using the following HSIC-IR p-value thresholds: (ONT-SVP) p-adj \(\le\) 0.05; (SR-SVP) p-adj \(\le\) 0.01. 150 genes shared by both ONT replicates are considered as ONT-SVP genes. (c) Gene-level SVP test agreements between ONT and SR. Left: One-sided chi-square test comparing SVP calling results. Right: SR-based HSIC-IR p-value distributions. Boxplots show median (center line), interquartile range (box), and 1.5× interquartile range (whiskers), with p-values from two-sided Wilcoxon rank-sum test. Negative background sample sizes vary as not all genes detected/tested in both platforms. (d) Left: Exon overlaps across platforms. Right: Xenium-based HSIC-IR p-value distributions in adult mouse brain. Boxplots show median (center line), interquartile range (box), and 1.5× interquartile range (whiskers). P-values from two-sided Wilcoxon rank-sum test. (e) Assessment of Xenium technical artifacts. Left: HSIC-IR adjusted p-values for non-gene features. Boxplots show median (center line), interquartile range (box), and 1.5× interquartile range (whiskers). Group distributions were compared using one-sided Kruskal-Wallis test. Y-axis was truncated (p-adj \(\ge\) 1e-40) for visualization purpose. Right: Spatial transcript density of selected genes. NegControlProbe_00041 variable usage is driven by codeword 5642, which shows a similar glial-cell-specific pattern as Bmp2-1345 and Trp53bp1-17048. (f) Scatter plots showing HSIC-IR adjusted p-values for genes detected in both Xenium Prime 5K and either Visium-ONT (left, Spearman’s ρ = 0.19) or Visium-SR (right, Spearman’s ρ = 0.15). Red dots indicate genes identified as SVP in the Visium platforms as in (g). Dashed red lines mark Xenium significance thresholds (p-adj = 2.5e-17). (g) Overlap of SVP genes across platforms. Events were identified using the following HSIC-IR p-value thresholds: (ONT-SVP) p-adj \(\le\) 0.05 in both replicates; (SR-SVP) p-adj \(\le\) 0.01; (Xenium-SVP) p-adj ≤ 2.5e-17.