A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

Abstract

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

Detecting ultralow-frequency mutations by Duplex Sequencing

“…Similarly, the genetic diversity found within microbial populations underlies their ability to adapt to changing environments, including development of drug resistance 10-13 , but this genetic diversity is difficult to directly assess owing to the high background error rate of conventional NGS sequencing. Other fields with a similar need for robust low-frequency mutation detection include forensics 14 , paleogenomics 15,16 , evolution 17 and toxicology 18 , as high-accuracy sequencing would allow one to assess the potential mutagenicity of new chemical compounds without the need for a genetic selection system to identify mutant genes.…”

The Application of Omics Technologies to the Study of Mammalian Toxicology

“…Identification of different patterns of mutagenesis may provide insight into causal mechanisms behind the mutagenic process [26,43,[132][133][134] These data suggested an odds ratio of approximately 26 for developing SJS in individuals that are carriers of the HLA-A*3101 allele. Considering the effect size of this variant, it would likely be of value clinically to perform genotyping of patients prior to the administration of carbamazepine in order to limit the risk of SJS.…”

“…Genomics approaches have also been used to identify mutation spectra signatures that are reflective of specific mutation processes elicited by chemical carcinogens [26]. In such studies, cancer genomes from individuals with different exposure histories (e.g., lung cancer from smokers and nonsmokers) are sequenced, and global patterns of mutagenesis (e.g., types of transitions, transversions, deletions, sequence context of mutations) are quantified.…”

“…It is well documented that carcinogens exhibit preferential reactivity with certain DNA bases, which then translates into specific distributions of mutation spectra (types of transition and transversion) which some have referred to as a 'mutation signature' [26,39,40]. Based on this observation, the sequencing of cancer genomes can -in theory -reveal mutational signatures that may be plausibly associated with the causal mutagenic agent [41,42].…”

Toxicogenomics and Cancer Susceptibility: Advances with Next-Generation Sequencing

“…Generally, there are two ways to perform toxicogenomics studies: (1) starting with conventional toxicological research and then focusing on omics approaches to detect systematic biological effects, and (2) starting with the omics study followed by conventional toxicological research to interrogate molecular mechanisms. NGS technologies have been used in both approaches [105][106][107][108].…”