To explicitly test if the CBS hotspots could be explained by the genome-wide elevated mutation rate at CBSs, we constructed a CBS-specific background mutation model. Since CBS mutation rates varied across tumor subtypes, this model further included the tumor subtype as a covariate. Also, since CBSs located at chromatin loop boundaries have higher somatic mutation burden than non-boundary CBSs15,18, the CBS-specific background model differentiated between CBSs inside and outside chromatin loop boundaries. CTCF loop domains have not been profiled in gastric tissue but tend to be cell-type invariant41,42. We therefore used a constitutive set of CTCF domains shared across three cell lines (CM12878, Jurkat, and K562) to define CTCF loop boundaries13,43. In addition, since the mutation spectrum at CBSs is distinct from the overall genomic mutation spectrum, we performed LASSO logistic regression to identify sequence context features correlated with the somatic mutation rate at CBSs. To identify other mutational processes that might be associated with the occurrence of CBS mutations, we calculated the correlation between the proportion of CBS mutations in each tumor and the percentage contribution of each COSMIC mutation signature to each tumor44. While CBS mutations are known to be positively associated with signature 1715, we found that CBS mutations were also strongly negatively associated with COSMIC mutation signature 1, an age related signature (Pearson correlation = −0.41; Supplementary Fig. 5). Therefore, we added the percentage contributions of mutation signatures 1 and 17 in each individual as covariates. Finally, this model also corrected for replication timing and local mutation rate. With this model, 9/11 CBS hotspots remained significant at the Bonferroni corrected significance threshold of 0.01 and the other two were borderline with adjusted P-values of 0.025 and 0.086 (Fig. 4g). Furthermore, seven additional CBSs became significant with the restricted hypothesis testing (Supplementary Table 4; Supplementary Figs. 6, 7). Mutations at these specific sites can therefore not be explained by a genome-wide elevated mutation rate at CBS, indicating that mutations at these focal sites are may be positively selected in gastric tumors.