We used this statistical framework to identify somatic mutation hotspots (both indels and point mutations) across the non-coding genome (Fig. 2b and Fig. 3a). The top indel hotspot was located ~100 kbp upstream of the AFDN gene, which is frequently translocated in leukemia and down-regulated in multiple cancer types34–36. The effect of hotspot mutations on AFDN expression could not be tested, as we lacked paired tumor expression data for the mutated samples. The second most significant indel hotspot was located in an intron of the PGC gene, which encodes the precursor of gastric proteinase pepsinogen (Supplementary Table 1). PGC is expressed at 11940 TPM in the stomach, 39 TPM in the lung, and ≤2 TPM in all other tissues in the Genotype-Tissue Expression (GTEx) project37,38. Interestingly, a recent study reported that LIPF, a lineage-specific gastric lipase, has broad enrichment of indels in GC39. Hypothesizing that other lineage-specific genes could show similar patterns of indel enrichment, we performed a gene-based recurrence analysis to identify all genes with broad enrichment of indels in their non-coding regions (combining promoter, untranslated, and intronic regions for each gene; Methods). Interestingly, the top three genes in this analysis were all lineage-specific genes highly expressed in stomach tissue: LIPF, PGC, and MUC6 (Fig. 2c; Supplementary Table 2). MUC6 encodes a mucin glycoprotein that is a major constituent of the gut mucosa, and is expressed at 133 TPM in stomach tissue, 38 TPM in the pancreas, and ≤2 TPM in all other tissues in GTEx37,38. However, consistent with the previous report39, non-coding indels in these three recurrently mutated lineage-specific genes were not associated with expression change (Fig. 2d–f).Fig. 3Genome-wide analysis of non-coding SNV hotspots. a The negative log P-values of SNV recurrence for all 21 bp regions genome-wide, only regions with at least three mutations are displayed. Significantly mutated hotspots overlapping CBSs are highlighted. The horizontal line marks the Bonferroni adjusted P-value of 0.01. b Log odds ratio of the enrichment of hotspot mutations and non-hotspot mutations in transcription factor binding regions and conserved regions. Error bars indicate the s.e.m of the log odds ratio. c Gastric cancer samples sorted by molecular subtype, with each row representing a significant mutation hotspot. Mutated samples are highlighted in black in the matrix. The mutation load of each sample is shown in the bottom panel. The right panel annotates the location of each hotspot with respect to annotated functional regions