Eunnara Cho , Carol D. Swartz , Andrew Williams , Miriam V. Rivas , Leslie Recio , Kristine L. Witt , Elizabeth K. Schmidt , Jeffry Yaplee , Thomas H. Smith , Phu Van , Fang Yin Lo , Charles C. Valentine III , Jesse J. Salk , Francesco Marchetti , Stephanie L. Smith-Roe , Carole L. Yauk
{"title":"纠错双工测序能够直接检测和定量人类TK6细胞的突变,具有很强的实验室间一致性","authors":"Eunnara Cho , Carol D. Swartz , Andrew Williams , Miriam V. Rivas , Leslie Recio , Kristine L. Witt , Elizabeth K. Schmidt , Jeffry Yaplee , Thomas H. Smith , Phu Van , Fang Yin Lo , Charles C. Valentine III , Jesse J. Salk , Francesco Marchetti , Stephanie L. Smith-Roe , Carole L. Yauk","doi":"10.1016/j.mrgentox.2023.503649","DOIUrl":null,"url":null,"abstract":"<div><p>Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, <em>N</em>-ethyl-<em>N</em>-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25–200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DS mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A significant increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (<em>r</em> = 0.97). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The consistent results across the three time points suggest that 48 h may be sufficient for mutation analysis post-exposure. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DS for characterizing chemical mutagenicity in both research and regulatory evaluation.</p></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":null,"pages":null},"PeriodicalIF":2.3000,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10395007/pdf/","citationCount":"0","resultStr":"{\"title\":\"Error-corrected duplex sequencing enables direct detection and quantification of mutations in human TK6 cells with strong inter-laboratory consistency\",\"authors\":\"Eunnara Cho , Carol D. Swartz , Andrew Williams , Miriam V. Rivas , Leslie Recio , Kristine L. Witt , Elizabeth K. Schmidt , Jeffry Yaplee , Thomas H. Smith , Phu Van , Fang Yin Lo , Charles C. Valentine III , Jesse J. Salk , Francesco Marchetti , Stephanie L. Smith-Roe , Carole L. Yauk\",\"doi\":\"10.1016/j.mrgentox.2023.503649\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, <em>N</em>-ethyl-<em>N</em>-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25–200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DS mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A significant increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (<em>r</em> = 0.97). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The consistent results across the three time points suggest that 48 h may be sufficient for mutation analysis post-exposure. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. 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Error-corrected duplex sequencing enables direct detection and quantification of mutations in human TK6 cells with strong inter-laboratory consistency
Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25–200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DS mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A significant increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (r = 0.97). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The consistent results across the three time points suggest that 48 h may be sufficient for mutation analysis post-exposure. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DS for characterizing chemical mutagenicity in both research and regulatory evaluation.
期刊介绍:
Mutation Research - Genetic Toxicology and Environmental Mutagenesis (MRGTEM) publishes papers advancing knowledge in the field of genetic toxicology. Papers are welcomed in the following areas:
New developments in genotoxicity testing of chemical agents (e.g. improvements in methodology of assay systems and interpretation of results).
Alternatives to and refinement of the use of animals in genotoxicity testing.
Nano-genotoxicology, the study of genotoxicity hazards and risks related to novel man-made nanomaterials.
Studies of epigenetic changes in relation to genotoxic effects.
The use of structure-activity relationships in predicting genotoxic effects.
The isolation and chemical characterization of novel environmental mutagens.
The measurement of genotoxic effects in human populations, when accompanied by quantitative measurements of environmental or occupational exposures.
The application of novel technologies for assessing the hazard and risks associated with genotoxic substances (e.g. OMICS or other high-throughput approaches to genotoxicity testing).
MRGTEM is now accepting submissions for a new section of the journal: Current Topics in Genotoxicity Testing, that will be dedicated to the discussion of current issues relating to design, interpretation and strategic use of genotoxicity tests. This section is envisaged to include discussions relating to the development of new international testing guidelines, but also to wider topics in the field. The evaluation of contrasting or opposing viewpoints is welcomed as long as the presentation is in accordance with the journal''s aims, scope, and policies.