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Jesse Falk

OpenTox 2022 Virtual Conferecne 

Next Generation Techniques for Genotoxicity and Carcinogenicity Prediction by Error-Corrected Duplex Sequencing

Current mutagenicity assays rely on indirect methods of detection that are limited to assessing short sequences of DNA in reporter genes. In addition to being cumbersome, and often highly dependent on specific model systems, the resulting data provides little-to-no mechanistic information about the nature of a mutagen, nor how it behaves across the genome. Duplex Sequencing (DS) is a highly generalizable mutation-detection technology that can directly detect ultra-rare mutations (on the order of 1-in-10-million) in any region of DNA of almost any tissue or cell type from any organism. DS yields high-fidelity mutagenicity data including mutation frequency (MF), mutation spectra and trinucleotide mutation signatures–the latter of which makes it possible to infer mutational processes that have been directly implicated in human cancers. Furthermore, DS is able to sensitively identify clonal expansions as early markers of carcinogenesis months in advance of overt tumor formation, even with non-mutagenic carcinogens. The presentation will provide an overview of this powerful emerging technology, along with examples of how DS is being used as a fit-for-purpose alternative approach in genetic toxicology to overcome current limitations in standard mutagenicity and carcinogenicity testing. In particular we will review recent data indicating how DS can support the 3Rs goals while improving upon current assays through: 1) rapid detection of mutagens in any rodent system,  thus eliminating the need for transgenic animals and increasing the feasibility of integration with existing acute toxicity assays to significantly reducing the number of animals used, 2) supporting new approach methodologies (NAMs) where mutagenesis assessment can be carried out in cells and/or organotypic cultures in lieu of animals, 3) assessing the off-target effects of gene editing and many other gene therapies which inherently must be carried out in human cells, not animals and, 4) applications for tracking early carcinogenesis via clonal expansions of cancer driver mutations to potentially reduce the need for animal-intensive 2 year rodent cancer bioassays.