Abstract
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.
Highlights
We present here a simple, phenotype-independent mutation assay using a PacBio RSII DNA sequencer employing single-molecule real-time (SMRT) sequencing technology
We previously reported a phenotypefree mutation assay using next-generation DNA sequencing [1]
The test strain Salmonella typhimurium YG7108, hisG46 rfa ΔuvrB bio adaST::kanr ogtST::catr, which is highly sensitive to alkylating agents, was used in this study [8]
Summary
The test strain Salmonella typhimurium YG7108, which is highly sensitive to alkylating agents, was treated with ENU (Fig. 2a) or its solvent DMSO, followed by dilution and growth overnight in LB medium to fix mutations. In the case of base substitutions, 19 and 160 mutations were called in the control and ENUtreated samples, respectively (Table 1). While these frequencies are consistent with the results of our previous study, they are still higher than the estimated values. The numbers of ‘real’ base substitution mutations were 0 and 132 in the control and ENU-treated samples, respectively (Table 4). Discussions In this paper, we successfully detected ultra-low frequency base substitution mutations by using a singlemolecule real-time sequencer with the SMRTbell strategy. This would reflect the presence of the DNA damages in the SMRTbell templates. Ongoing improvements to the hardware and software of the SMRT sequencer and to the bioinformatics of mutation detection will likely overcome this problem in the near future
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