Hawk-Seq™ differentiates between various mutations in <i>Salmonella typhimurium</i> TA100 strain caused by exposure to Ames test-positive mutagens

Otsubo, Yasufumi; Matsumura, Shoji; Ikeda, Naohiro; Morita, Osamu

doi:10.1093/mutage/geab006

Cited by 8 publications

(6 citation statements)

References 44 publications

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“…Results from a variety of assays have shown that the background MFs of many in vitro and in vivo toxicological models are ≥1 × 10 −7 mut/bp (Masumura et al, 2016; Otsubo et al, 2021; Valentine 3rd et al, 2020; Wang et al, 2021). Although the limit of detection of PacBio HiFi has yet to be determined, the fact that it consistently recorded MFs ~1 × 10 −7 mut/bp in unexposed samples suggests that its sensitivity is sufficient to detect the effects of mutagens weaker than EMS.…”

Section: Discussionmentioning

confidence: 99%

Genome‐wide detection of ultralow‐frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms by PacBio sequencing

Miranda

Alund

Yan

et al. 2022

Environ and Mol Mutagen

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Many conventional genetic toxicology assays require specialized cell cultures or animals and can only detect mutations that inactivate the function of a reporter gene. These limitations make such assays incompatible with many toxicological models but could be overcome by the development of techniques capable of directly detecting genome‐wide somatic mutations through DNA sequencing. PacBio sequencing can generate almost error‐free consensus reads by repeatedly inspecting both DNA strands from circularized molecules (a method known as PacBio HiFi). In this study, we show that PacBio HiFi can detect genome‐wide ultralow‐frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms. The mutation frequencies (MFs) of unexposed samples in both models were ~1 × 10−7 mutations per base pair. Compared to these controls, PacBio HiFi detected MF increases of 23‐fold in cultures of L5178Y cells exposed to 5 mM ethyl methanosulfonate (EMS) for 4 h, and 5‐, 12‐, and 29‐fold in cultures of C. elegans worms exposed to 12.5, 25, and 50 mM EMS for 4 h, respectively. In both models, the mutation spectra of controls were diverse, while those derived from EMS‐exposed samples were dominated by C:G → T:A transitions. To validate these results, clone sequencing analyses were performed on the same cultures of L5178Y cells. The results obtained by clone sequencing and PacBio HiFi were almost identical. Our results suggest that PacBio sequencing could be used for the detection, quantitation, and characterization of mutations in any DNA‐containing sample, including those that are not compatible with conventional mutation detection approaches.

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Section: Discussionmentioning

confidence: 99%

Genome‐wide detection of ultralow‐frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms by PacBio sequencing

Miranda

Alund

Yan

et al. 2022

Environ and Mol Mutagen

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“…Genomic DNA samples of TA100, which were exposed to 1000 µg/tube of 3-methylcholanthrene (3MC), 1000 µg/tube of 7,12-dimethylbenz[a]anthracene (DMBA), and solvent control (DMSO) under suspension culture conditions, were used (Otsubo et al 2021 ). First, 60–120 ng of TA100 genomic DNA samples were sheared to fragments with a peak size of 350 bp using a sonicator (Covaris, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…and demonstrated its use in evaluating chemical mutagenicity and carcinogenicity (Matsumura et al 2019;Otsubo et al 2021).…”

Section: Introductionmentioning

confidence: 99%

“…Because DNA damage exists on only one strand of the dsDNA fragment, DCSSs can dramatically improve sequencing accuracy (10 −7 –10 −8 bp) by utilizing sequence information from both strands of dsDNA. We have developed a DCSS called Hawk-Seq™ and demonstrated its use in evaluating chemical mutagenicity and carcinogenicity (Matsumura et al 2019 ; Otsubo et al 2021 ).…”

Section: Introductionmentioning

confidence: 99%

“…Using Hawk-Seq™ analysis, we found that the errors on G:C base pairs occurred approximately five times more frequently than those on A:T base pairs. These errors could hamper the detection or characterization of extremely rare mutations (Otsubo et al 2021 ). Some have suggested that these errors are attributable to oxidized guanine located on single-strand (SS) overhangs, which may arise at the ends of sonicated DNA fragments.…”

Section: Introductionmentioning

confidence: 99%

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Single-strand specific nuclease enhances accuracy of error-corrected sequencing and improves rare mutation-detection sensitivity

et al. 2021

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Error-corrected sequences (ECSs) that utilize double-stranded DNA sequences are useful in detecting mutagen-induced mutations. However, relatively higher frequencies of G:C > T:A (1 × 10−7 bp) and G:C > C:G (2 × 10−7 bp) errors decrease the accuracy of detection of rare G:C mutations (approximately 10−7 bp). Oxidized guanines in single-strand (SS) overhangs generated after shearing could serve as the source of these errors. To remove these errors, we first computationally discarded up to 20 read bases corresponding to the ends of the DNA fragments. Error frequencies decreased proportionately with trimming length; however, the results indicated that they were not sufficiently removed. To efficiently remove SS overhangs, we evaluated three mechanistically distinct SS-specific nucleases (S1 Nuclease, mung bean nuclease, and RecJf exonuclease) and found that they were more efficient than computational trimming. Consequently, we established Jade-Seq™, an ECS protocol with S1 Nuclease treatment, which reduced G:C > T:A and G:C > C:G errors to 0.50 × 10−7 bp and 0.12 × 10−7 bp, respectively. This was probably because S1 Nuclease removed SS regions, such as gaps and nicks, depending on its wide substrate specificity. Subsequently, we evaluated the mutation-detection sensitivity of Jade-Seq™ using DNA samples from TA100 cells exposed to 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene, which contained the rare G:C > T:A mutation (i.e., 2 × 10−7 bp). Fold changes of G:C > T:A compared to the vehicle control were 1.2- and 1.3-times higher than those of samples without S1 Nuclease treatment, respectively. These findings indicate the potential of Jade-Seq™ for detecting rare mutations and determining the mutagenicity of environmental mutagens.

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Genotoxicity testing and recent advances

Luan

Honma

2021

GENOME INSTAB. DIS.

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Hawk-Seq™ differentiates between various mutations in Salmonella typhimurium TA100 strain caused by exposure to Ames test-positive mutagens

Cited by 8 publications

References 44 publications

Genome‐wide detection of ultralow‐frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms by PacBio sequencing

Genome‐wide detection of ultralow‐frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms by PacBio sequencing

Single-strand specific nuclease enhances accuracy of error-corrected sequencing and improves rare mutation-detection sensitivity

Genotoxicity testing and recent advances

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