Direct Quantification of in vivo Mutagenesis Using Duplex Sequencing

The ability to accurately measure mutations is critical for both basic research and in the identification of potential drug and chemical carcinogens. Current methods for in vivo quantification of drug and chemical mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and do not fully represent other species such as humans. Next generation sequencing (NGS) is a conceptually attractive alternative for massively parallel mutation detection in the DNA of any organism, however, the limits of detection for standard NGS are poor. Technical error rates (~1x10-3) of NGS obscure the true abundance of somatic mutations, which can exist at frequencies of 1x10-7 or less. Using Duplex Sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by 3 carcinogens in 5 tissues of 2 strains of mice within 31 days following exposure. We observed a strong correlation between mutation induction measured by Duplex Sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each tested compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. In addition to detecting mutagenesis, we identified clear and early signs of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by apparent clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis, as well as early clonal evolutionary hallmarks of carcinogenesis. Duplex Sequencing can be broadly applied to chemical safety testing, basic mutational research, and related clinical uses.