Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli
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 Escher...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
eLife Sciences Publications Ltd
2022-10-01
|
| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/83780 |
| _version_ | 1811329520176726016 |
|---|---|
| author | Hidehiko Kawai Ren Iwata Shungo Ebi Ryusei Sugihara Shogo Masuda Chiho Fujiwara Shingo Kimura Hiroyuki Kamiya |
| author_facet | Hidehiko Kawai Ren Iwata Shungo Ebi Ryusei Sugihara Shogo Masuda Chiho Fujiwara Shingo Kimura Hiroyuki Kamiya |
| author_sort | Hidehiko Kawai |
| collection | DOAJ |
| description | 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 ultraviolet (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. |
| first_indexed | 2024-04-13T15:45:27Z |
| format | Article |
| id | doaj.art-83a432033f844ff6b05ea8819b890fc1 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-13T15:45:27Z |
| publishDate | 2022-10-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-83a432033f844ff6b05ea8819b890fc12022-12-22T02:41:00ZengeLife Sciences Publications LtdeLife2050-084X2022-10-011110.7554/eLife.83780Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coliHidehiko Kawai0https://orcid.org/0000-0003-2213-7166Ren Iwata1Shungo Ebi2Ryusei Sugihara3Shogo Masuda4Chiho Fujiwara5Shingo Kimura6Hiroyuki Kamiya7https://orcid.org/0000-0001-6866-5322Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; School of Pharmaceutical Sciences, Hiroshima University, Hiroshima, JapanSchool of Pharmaceutical Sciences, Hiroshima University, Hiroshima, JapanSchool of Pharmaceutical Sciences, Hiroshima University, Hiroshima, JapanSchool of Pharmaceutical Sciences, Hiroshima University, Hiroshima, JapanGraduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, JapanSchool of Pharmaceutical Sciences, Hiroshima University, Hiroshima, JapanAnalysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, JapanGraduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; School of Pharmaceutical Sciences, Hiroshima University, Hiroshima, JapanA 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 ultraviolet (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.https://elifesciences.org/articles/83780mutagenesis assayNGSsupFUV mutagenesis |
| spellingShingle | Hidehiko Kawai Ren Iwata Shungo Ebi Ryusei Sugihara Shogo Masuda Chiho Fujiwara Shingo Kimura Hiroyuki Kamiya Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli eLife mutagenesis assay NGS supF UV mutagenesis |
| title | Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli |
| title_full | Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli |
| title_fullStr | Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli |
| title_full_unstemmed | Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli |
| title_short | Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli |
| title_sort | development of a versatile high throughput mutagenesis assay with multiplexed short read ngs using dna barcoded supf shuttle vector library amplified in e coli |
| topic | mutagenesis assay NGS supF UV mutagenesis |
| url | https://elifesciences.org/articles/83780 |
| work_keys_str_mv | AT hidehikokawai developmentofaversatilehighthroughputmutagenesisassaywithmultiplexedshortreadngsusingdnabarcodedsupfshuttlevectorlibraryamplifiedinecoli AT reniwata developmentofaversatilehighthroughputmutagenesisassaywithmultiplexedshortreadngsusingdnabarcodedsupfshuttlevectorlibraryamplifiedinecoli AT shungoebi developmentofaversatilehighthroughputmutagenesisassaywithmultiplexedshortreadngsusingdnabarcodedsupfshuttlevectorlibraryamplifiedinecoli AT ryuseisugihara developmentofaversatilehighthroughputmutagenesisassaywithmultiplexedshortreadngsusingdnabarcodedsupfshuttlevectorlibraryamplifiedinecoli AT shogomasuda developmentofaversatilehighthroughputmutagenesisassaywithmultiplexedshortreadngsusingdnabarcodedsupfshuttlevectorlibraryamplifiedinecoli AT chihofujiwara developmentofaversatilehighthroughputmutagenesisassaywithmultiplexedshortreadngsusingdnabarcodedsupfshuttlevectorlibraryamplifiedinecoli AT shingokimura developmentofaversatilehighthroughputmutagenesisassaywithmultiplexedshortreadngsusingdnabarcodedsupfshuttlevectorlibraryamplifiedinecoli AT hiroyukikamiya developmentofaversatilehighthroughputmutagenesisassaywithmultiplexedshortreadngsusingdnabarcodedsupfshuttlevectorlibraryamplifiedinecoli |