Extended Data Fig. 2: Spatial genome alignment on sequential 5 kb M-DNA-FISH imaging of mESC Sox2 locus.

(a) Median distance matrix of spatial genome alignment resolved Sox2 locus (chr3:34601078-34811078), sequentially imaged at a finer 5 kb resolution, using a different imaging protocol based on multiplexed DNA-FISH. (b) Median distance matrix identified by traditional spot selection using an expectation-maximization routine previously used to analyze M-DNA-FISH datasets. (c) KR-normalized Hi-C contact matrix of the Sox2 locus, juxtaposed as a comparison to median distance matrices discovered by two chromatin tracing algorithms. (d, e) Correlation plot of pairwise genomic distances discovered by spatial genome alignment and an E-M spot selection routine respectively, plotted against KR normalized Hi-C contact frequency. (f) Heatmaps of tabulated distances (radius = 150 nm) from spatial genome alignment of M-DNA-FISH imaging (bottom left) juxtaposed to contact frequencies from Hi-C binned at 5 kb (top right). (g) Heatmaps of tabulated distances (radius = 150 nm) from E-M spot selection of M-DNA-FISH imaging (bottom left) juxtaposed to contact frequencies from Hi-C binned at 5 kb (top right). (h) Spearman correlation (y-axis) of tabulated distances from M-DNA-FISH tracing against bulk Hi-C, across a range of distance thresholds (x-axis) for tabulating distances into contacts, delineated for each of the tracing methods (spatial genome alignment and E-M spot selection).