We then performed a genome-wide analysis of SNVs in non-coding regions and identified 34 significant mutation hotspots (Bonferroni adjusted P-value < 0.01; Fig. 3a; Supplementary Table 3). These hotspots were enriched in conserved sequences and TF binding regions, suggesting that many hotspot mutations may disrupt functional elements (Fig. 3b). Strikingly, of the 34 mutation hotspots, 11 were located in CBSs (Fig. 3a, c). The majority of mutations at CBS hotspots occurred in CIN tumors (71%, P = 0.012 by two-sided Fisher’s exact test), which is the most common GC subtype, accounting for ~50% of all GC cases (Fig. 3c). The remaining 23 non-CBS hotspots often overlapped gene regions, but never co-located with TF binding regions. Furthermore, we observed a depletion of somatic mutations at gastric-specific TFBSs among the non-hotspot mutations (Fig. 3b). Overall, gastric tissue TFBSs comprises about 1% of the genome, but only 0.58% of the non-hotspot mutations were located in these regions. A similar depletion of mutations was observed for constitutive TFBSs (Supplementary Fig. 3). This is striking, as two recent studies have found that somatic mutation rates are elevated at transcription factor binding sites (TFBSs), and that this higher overall mutation load at TFBSs may be explained by reduced accessibility to nucleotide-excision repair (NER) enzymes at these sites16,17. This phenomenon is primarily observed in melanoma and lung adenocarcinoma where NER plays an important role in repairing carcinogen induced DNA lesions16. In contrast, our finding demonstrates that NER and TF occupancy is not a cause of regional mutational bias in GC.