Development of a versatile high-throughput mutagenesis assay with multiplexed short read NGS using DNA-barcoded supF shuttle vector library amplified in non-SOS E. coli
- Hiroshima University, Japan
Abstract
A forward mutagenesis assay using the supF gene has been widely employed for the last several decades in studies addressing mutation frequencies and mutation spectra associated with various intrinsic and environmental mutagens. In this study, by using a supF shuttle vector and non-SOS-induced Escherichia coli with short read Next Generation Sequencing (NGS) technology, we present an advanced method for the study of mutations, which is simple, versatile, and cost-effective. We demonstrate the performance of our newly developed assay via pilot experiments with UV-irradiation, the results from which emerge more relevant than expected. The NGS data obtained from samples of the indicator E. coli grown on titer plates provides mutation frequency and spectrum data, and uncovers obscure mutations that cannot be detected by a conventional supF assay. Furthermore, a very small amount of NGS data from selection plates reveals the almost full spectrum of mutations in each specimen and offers us a novel insight into the mechanisms of mutagenesis, despite them being considered already well-known. We believe that the method presented here will contribute to future opportunities for research on mutagenesis, DNA repair, and cancer.
Data availability
NGS raw data has been uploaded to DDBJ database (http://www.ddbj.nig. ac.jp) under the accession number PRJDB13753 (DRA014420 for Figures 2-4, DRA014421 for Figures 5 and 6, DRA014422 for Figures 7 and 8, DRA014422 for the samples without transformation into E. coli)
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Author details
Funding
Japan Society for the Promotion of Science (JP 19K123222)
- Hidehiko Kawai
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Kawai et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Development of a versatile high-throughput mutagenesis assay with multiplexed short read NGS using DNA-barcoded supF shuttle vector library amplified in non-SOS E. coli
eLife 11:e83780.
https://doi.org/10.7554/eLife.83780
