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Error-corrected Duplex Sequencing enables direct detection and quantification of mutations in human TK6 cells with remarkable inter-laboratory consistency

Authors :
Eunnara Cho
Carol D. Swartz
Andrew Williams
Miriam Rivas
Leslie Recio
Kristine L. Witt
Elizabeth K. Schmidt
Jeffry Yaplee
Thomas H. Smith
Phu Van
Fang Yin Lo
Charles C. Valentine
Jesse J. Salk
Francesco Marchetti
Stephanie L. Smith-Roe
Carole L. Yauk
Publication Year :
2023
Publisher :
Cold Spring Harbor Laboratory, 2023.

Abstract

Error-corrected Duplex Sequencing (DuplexSeq) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DuplexSeq 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 DuplexSeq mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A robust 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 (R2=0.95). 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 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 DuplexSeq for characterizing chemical mutagenicity in both research and regulatory evaluation.

Details

Database :
OpenAIRE
Accession number :
edsair.doi...........0297e7108e98c4ad7df2f12bf5adc5ad