Supplementary Data to: Mutational signature analysis of Asian OSCCs reveals novel mutational signature with exceptional sequence context specificity by Boot et al. Background Study of mutational signatures in cancers can reveal the mutagenic processes that cells were exposed to prior and during tumourigenesis. Although recent work has identified 65 distinct single nucleotide substitution signatures, we expect that as-yet-undiscovered mutational processes will be able to shed further light on mutagenesis leading to carcinogenesis. Methods We analyzed the mutational spectra of 36 Asian oral squamous cell carcinomas. Results The mutational spectra of most of the carcinomas could be explained by known mutational signatures, but one sample showed a novel mutational signature. Whole-genome sequencing revealed that this mutational signature was characterized by a preponderance of thymine mutations, strong transcriptional strand bias, and a striking enrichment for adenines in the 4 base pairs 5’ of mutation sites. The enrichment was strongest 3 base pairs 5' of mutated thymines, where 93.5% of bases were adenines. We also observed deletions of single thymines with similar 5’ enrichment for adenines both inside and outside of thymine repeats. Examination of publicly available whole-genome and whole-exome sequencing data of 23,829 tumours revealed this signature in 23 tumours from sites including the bile-duct, bladder, pancreas and rectum. Conclusions Although the aetiology of this mutational signature remains unknown, the transcriptional strand bias suggests adduct formation on adenines. We speculate that the signature may have been caused by duocarmycins. These are naturally occurring large DNA-crosslinking molecules produced by some Streptomyces bacteria, and which were also used in the past in clinical trials. We call this novel mutational signature SBS_AnT.