Extended Data Fig. 4 ∣. Evaluation of pre-QC cell clustering results from the TOPACIO dataset.

a, Spatial distribution of unclustered cells (green dots) from the pre-QC TOPACIO embedding (Fig. 3a) as shown in sample 55 demonstrating no discernable pattern of sampling bias; Hoechst (gray) shown for reference. b, Silhouette scores for pre-QC TOPACIO clusters shown in (Fig. 3a). c, Stacked bar charts showing the relative contribution of each patient sample to each cluster. Inset shows more detailed views for 48 of the 492 clusters that were selected at random for curating image patch galleries for visual inspection of cells comprising these clusters (Online Supplementary Fig. 2). d, Line plot showing cell counts per TOPACIO cluster. Clusters with cell counts below the horizonal dashed red line are those with fewer than 3K cells which are highlighted in the TOPACIO embedding (inset) by red scatter points at their relative positions. e, TOPACIO embedding colored by meta-clusters shown in Fig. 3b. f, TOPACIO sample 55 at low (left) and high (right) magnification showing Hoechst signals for the first three imaging cycles: 1 (green), 2 (red), and, 3 (blue) superimposed to demonstrate cross-cycle image alignment errors in this sample. Small white box at the bottom-right of the low magnification image shows the location of the higher magnification image. White dots in the image magnification image highlight cluster 15 cells which have formed due to this image alignment artefact.