Extended Data Fig. 5: Single cell measurement of chromosomal instability.
From: Ongoing genome doubling shapes evolvability and immunity in ovarian cancer
a. Schematic of nearest neighbor difference (NND) using fraction of the genome different as a distance measure (left). Shown are two pairs of example nearest nearest neighbor cells and regions of the genome that are different for a 0×WGD cell (middle) and a 1×WGD cell (right). Each point is a 500 kb bin colored by the assigned copy-number state, and the y-axis shows ploidy-scaled GC-normalized read counts. b. Empirical distribution of NND for all cells, and beta distribution fit (red). c. NND (y-axis) by ploidy (x-axis) for cells from patient OV-081. Color indicates divergent status, and WGD multiplicity for non-divergent cells. d. Copy-number profiles for example 0×WGD (top), 1×WGD (middle) and divergent (bottom) cells from patient OV-081. Arrows highlight homozygously deleted regions. e. Arm nullisomy rates (counts per cell) for divergent and non-divergent cells in WGD-low and WGD-high tumors. Shown is the distribution of mean rates per population in each patient (only those populations with at least 10 cells are included): WGD-low non-divergent n = 14, WGD-low divergent n = 6, WGD-high non-divergent n = 20, WGD-high divergent n = 12 populations. Mann-Whitney U one-sided test significance is annotated as ‘ns’: 5.0 × 10−2 < p <= 1.0, ‘*’: 1.0 × 10−2 < p <= 5.0 × 10−2, ‘**’: 1.0 × 10−3 < p <= 1.0 × 10−2, ‘***’: 1.0 × 10−4 < p <= 1.0 × 10−3, ‘****’: p <= 1.0 × 10−4. Center line shows the median, box boundaries show quartiles, and whiskers indicate 1.5×IQR. WGD-low p = 2.6 × 10−5, WGD-high p = 2.0 × 10−6. f. Boxplots comparing fraction of divergent cells between WGD multiplicity populations for WGD-low and WGD-high tumors (only those populations with over 20 cells are included): WGD-low 0xWGD n = 14, WGD-low 1xWGD n = 8, WGD-high 0xWGD n = 2, WGD-high 1xWGD n = 25, WGD-high 2xWGD n = 4 populations. Mann-Whitney U one-sided test significance is annotated as per e. Boxplots are defined as per e. WGD-low (0xWGD vs 1xWGD) p = 8.2 × 10−4, WGD-high (1xWGD vs 2xWGD) p = 8.4 × 10−5. g. Fraction of divergent cells (y-axis) by age of the WGD as measured by C > T CpG mutations gained since WGD (x-axis). Shown is the p-value of a two-sided Spearman correlation after removing the three patients with the oldest WGDs. Two-sided Spearman correlation retaining these outliers is ρ = −0.47 p = 0.019. Shaded region indicates 95% confidence interval. h. MEDICC2 phylogeny (left) total copy number (center) and inferred cell-specific copy number changes (right) for patient OV-110. i. Coefficients, 95% confidence intervals, and p-values for the WGD term of a GEE model of chromosome, arm and segment loss and gain rates (counts per cell, normalized for genome size) for n = 54 adnexa vs non-adnexa subpopulations from the 37 patients with event rate estimates. The GEE model includes patient age, WGD status (high vs low), mutation signature (FBI vs non-FBI) and site (Adnexa vs non-Adnexa). j. Example immunofluorescence images of WGD-high and WGD-low tumor samples with varying MN rates. Images are annotated with the slide-level MN rates, calculated as the median MN rate across all tumor ROI regions within the slide. Top panels: Multi-channel overlay images of DAPI, cGAS and panCK intensity at high magnification. Bottom panels: Segmentation masks for cGAS+ MN and PN, including examples of micronuclei with annotated area size in μm2. k. Ratio of losses to gains for chromosomes (left) and chromosome arms (right). Cell specific refers to changes on leaf branches of the MEDICC2 phylogeny which are split by WGD-low and WGD-high tumor type. Ancestral changes are split into non-WGD, pre-WGD, and post-WGD as defined for Fig. 4B. Each datapoint for the cell specific distributions is a ratio of losses to gains for a single patient. Each datapoint for non-WGD, pre-WGD, and post-WGD distributions is a ratio computed from the root branch of the MEDICC2 phylogeny for a patient, distinguishing pre- and post-WGD changes for WGD-high tumors and including all changes for WGD-low tumors. Error bars indicate 95% confidence interval. Patients OV-045 and OV-025 with multiple parallel WGD events were excluded from this analysis. Mann-Whitney one-sided multiple-hypothesis-corrected U test p < 1.3 × 10−3 for chromosome event ratios, p = 0.016 for WGD-low vs. non-WGD arm event ratios, and p < 7.1* × 10−3 for remaining arm comparisons. WGD-low n=non-WGD n = 14, WGD-high n = 27, and pre-WGD n=post-WGD n = 21. l. Number of post-WGD chromosome and arm gains and losses (x-axis) compared to the mutation time in C > T CpG counts (y-axis) measured since the WGD event. Spearman correlation coefficients and p-values are shown.
