Extended Data Fig. 3: The frequency of SCEs correlates with mutation rate, and localizing reference genome assembly errors.

a, The relationship between single nucleotide substitution mutation load and detected SCE events in C3H tumours. DEN is known to produce ethyl aducts on the sugar-phosphate backbone of DNA as well as mutation-inducing modifications to the bases¹⁰ which could lead to strand breaks⁶⁰ triggering SCE. The frequent observation and correlation between rates of SCE and point mutation supports this view. Counts of SCE (y-axis) are based on down-sampling to 10,000 informative mutations per tumour to ensure equal power to detect SCE in each tumour. Tumours with <50% cellularity (pink) have high mutation load and form a sub-group with few detected SCE events; these are suspected to be polyclonal tumours and were excluded from the Pearson’s correlation reported (n = 335 independent tumour samples, implemented in a two-sided test, significance from Fisher’s transform). b, As for a, but showing CAST derived tumours (n = 84, after cellularity exclusions n = 77). c, Evaluation of the relationship between mutation load and ability to detect SCE events. Mutations from C3H tumour 94315_N8 (shown in Fig. 2) randomly down-sampled and segmentation analysis applied. The y-axis shows the percentage of SCE events detected (100 replicates, mean red, 95% C.I. pink). The x-axis is on a log-scale: 95% of C3H and >95% of CAST tumours have mutation counts to the right of the blue vertical line. Down-sampling other tumours gave comparable results. d, The same down-sampling data as shown in c but the y-axis shows the percent of mutations with the correct (same as full data) mutational asymmetry assignment (mean red, 95% C.I. pink). e, Candidate C3H reference genome assembly errors. Genome coordinates shown on the x-axis. Immediate switches between Watson and Crick asymmetry are not expected on autosomes unless both copies of the chromosome have a SCE event at equivalent sites. However, inversions and translocations between the sequenced genomes and the reference assembly are expected to produce immediate asymmetry switches. The discordant segment coverage count (black y-axis) shows the number of informative tumours (those with either Watson or Crick strand asymmetry at the corresponding genome position) that suggest a tumour genome to reference genome discrepancy. Consensus support (brown y-axis) plotted as triangles shows the percentage of informative tumours that support a genomic discrepancy at the indicated position (only shown for values >50% support). The two sites on chromosome 6 in C3H correspond to a previously identified C3H strain specific inversion that is known to be incorrectly oriented in the C3H reference assembly⁵⁴. f, Candidate CAST reference genome assembly errors, plotted as per e. The candidate misassembly on chromosome 14 in both strains occurs at an approximately orthologous position, suggesting a rearrangement shared between strains or a misassembly in the BL6 GRCm38 reference assembly against which other mouse reference genome assemblies have been scaffolded.