Extended Data Fig. 1: Overview of the analyses provided in this study.

a, The overall results generated in this work derive from four independent investigations of the colibactin-induced double-strand breaks (DSBs), using sBLISS technology. The resulting data sets were first used to identify the colibactin damage motif (CDM), which turned out to be best described by the consensus hexameric sequence AAWWTT. b, Topological analyses and the assignment of shape characteristics led to a virtual model of colibactin–DNA interaction, which is compatible with a colibactin-induced inter-strand cross-link formation between two adenines at positions 2(+) and 5(-) of the minor groove. c, The detection of the CDM served to identify associated single nucleotide substitutions in large human cancer genome data sets - both exome (TCGA) and whole genome (Finish cohort). The comparison of different cancer entities indicated notable enrichment of CDM-associated mutations in some colorectal cancer. The comparison of colon cancer types indicated an enrichment at distal colon locations. The classification of CDM-associated mutations revealed a strong correlation with known triplet signatures, above all with the signature SBSA, which was previously identified in normal colon epithelia. d, An independent analysis of the exact DSB endpoints led to the identification of putative processing sites following colibactin-induced DNA damage. Accordingly, the processing sites were located downstream (in a 5’>3’ direction) of the putatively-alkylated adenines on opposite strands, generating 2 nt long 5’ overhangs at the broken DNA. Alignment of these break positions with the location of colibactin-specific cancer mutations revealed a striking local correspondence, indicating the occurrence of mutations at distinct sites on the CDM. e, Overall, the sBLISS-derived analyses provide extensive insight into the mutational function of colibactin and reveal strong links to human cancer.