Differentiating epigenetic marks and DNA adducts at the KRAS codon 12 mutation hotspot using ONT/ELIGOS sequencing

Mutations-whether inherited or acquired-are central to cancer development, and reducing mutagenesis is an effective strategy for lowering cancer rates. At its simplest, DNA mutations arise from error-prone DNA replication or from the replication of DNA following incorrect repair of DNA lesions. Mechanistically, mutations occur when polymerases erroneously replicate DNA, with error rates increasing significantly in the presence of DNA modifications or DNA adducts. Among DNA modifications, epigenetic marks such as 5-methyl-deoxycytidine (5mdC) are the most abundant, occurring at a frequency of approximately one methylated cytosine per 100 cytosines. This is substantially higher than the frequency of endogenous DNA adducts (about one in 10,000 nucleotides) and exogenous DNA adducts (often less than one in 100 million normal nucleotides). Therefore, it remains unclear whether a specific mutation arises from errors in epigenetic processes, or from endogenous- and exposure-induced DNA adducts. Chemically specific methods for DNA adduct detection, unfortunately, often do not provide information about their position within the genome, prohibiting identification of the DNA adduct causing specific mutations. To address these knowledge gap, our group developed a nanopore-based DNA sequencing approach, called “ELIGOS” (Oxford Nanopore Technology-Epitranscriptional/ Epigenomical Landscape Inferring from Glitches of ONT Signals) that can (i) detect and (ii) differentiate between various types of epigenic marks and exposure-induced DNA adducts at base pair resolution while sequencing the DNA. ONT/ELIGOS provides an innovative tool to study the DNA lesions and mutations in pre-selected DNA regions. In this work, we present our latest ONT/ELIGOS findings demonstrating the detection and identification of DNA adducts at codon 12 within the KRAS cancer driver gene. In addition to common epigenetic marks and exposure-derived DNA adducts, our current panel of DNA adducts includes common endogenous lesions. Together these novel approaches can reveal the dynamics changes in single DNA molecules.