A Pilot Study for the Mutation Assay Using a High-throughput DNA Sequencer

Matsuda, Tomonari; Takamune, Makiko; Matsuda, Yoko; Yamada, Masami

doi:10.3123/jemsge.35.53

Cited by 10 publications

(10 citation statements)

References 3 publications

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“…These techniques involve the sequences of both original dsDNA strands and enable the accurate detection of true mutations, after they are distinguished from sequencing errors. The sequence accuracy of these techniques reportedly proved to be sufficient for the detection of mutations induced by chemical exposure (Matsuda et al 2013;Chawanthayatham et al 2017). However, most existing methods require either additional molecular barcodes during library preparation to discriminate between molecules or a specific sequence apparatus that has been used only in a few studies.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide somatic mutation analysis via Hawk-Seq™ reveals mutation profiles associated with chemical mutagens

et al. 2019

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It is difficult to identify mutagen-induced genome-wide somatic mutations using next generation sequencing; hence, mutagenic features of each mutagen and their roles in cancer development require further elucidation. We described Hawk-Seq™, a highly accurate genome sequencing method and the optimal conditions, for using it to construct libraries that would enable the accurate (c.a. 1 error/10 7-10 8 bp) and efficient survey of genome-wide mutations. Genomic mutations in gpt delta mice or Salmonella typhimurium TA100 exposed to methylnitrosourea (MNU), ethylnitrosourea (ENU), diethylnitrosamine (DEN), benzo[a]pyrene (BP), and aristolochic acid (AA) were profiled using Hawk-Seq™ to analyse positions, substitution patterns, or frequencies. The resultant vast mutation data provided high-resolution mutational signatures, including for minor mutational fractions (e.g. G:C>A:T by AA), which enabled the clarification of the mutagenic features of all mutagens. The 96-type mutational signatures of MNU, AA, and BP indicate their partial similarity to signature 11, 22, and 4 or 29, respectively. Meanwhile, signatures attributable to ENU and DEN were highly similar to each other, but not to signature 11, suggesting that the mechanisms of these agents differed from those of typical alkylating agents. Thus, Hawk-Seq™ can clarify genome-wide chemical mutagenicity profiles at extraordinary resolutions, thereby providing insight into mutagen mechanisms and their roles in cancer development.

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Section: Introductionmentioning

confidence: 99%

Genome-wide somatic mutation analysis via Hawk-Seq™ reveals mutation profiles associated with chemical mutagens

et al. 2019

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“…Although many mutation assays have been developed, most rely on some kind of phenotypic selection, which involves time-consuming procedures and is potentially biased. We previously reported a phenotype-free mutation assay using next-generation DNA sequencing [ 1 ]. In that study, we treated a Salmonella typhimurium strain with a mutagen to induced and fix mutations, followed by colony isolation and whole-genome sequencing of the colonies.…”

Section: Introductionmentioning

confidence: 99%

Mutation assay using single-molecule real-time (SMRTTM) sequencing technology

Matsuda

Yamada

2015

Genes and Environ

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IntroductionWe present here a simple, phenotype-independent mutation assay using a PacBio RSII DNA sequencer employing single-molecule real-time (SMRT) sequencing technology. Salmonella typhimurium YG7108 was treated with the alkylating agent N-ethyl-N-nitrosourea (ENU) and grown though several generations to fix the induced mutations, the DNA was extracted and the mutations were analyzed by using the SMRT DNA sequencer.ResultsThe ENU-induced base-substitution frequency was 15.4 per Megabase pair, which is highly consistent with our previous results based on colony isolation and next-generation sequencing. The induced mutation spectrum (95% G:C → A:T, 5% A:T → G:C) is also consistent with the known ENU signature. The base-substitution frequency of the control was calculated to be less than 0.12 per Megabase pair. A current limitation of the approach is the high frequency of artifactual insertion and deletion mutations it detects.ConclusionsUltra-low frequency base-substitution mutations can be detected directly by using the SMRT DNA sequencer, and this technology provides a phenotype-independent mutation assay.

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“…The hisG + colonies were randomly isolated, and genomic DNA was extracted using a Puregene Cell and Tissue kit (Qiagen). The mutational profiles induced by haloalkanes were subsequently examined using whole-genome sequencing with reference to a previous report ( 18 ). Using 3 µg of DNA and the SureSelect XT Library Prep Kit (Agilent Technologies), we prepared whole-genome sequencing libraries according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%