Mutation assay using single-molecule real-time (SMRTTM) sequencing technology
Open Access
- 1 September 2015
- journal article
- research article
- Published by Springer Science and Business Media LLC in Genes and Environment
- Vol. 37 (1), 1-10
- https://doi.org/10.1186/s41021-015-0017-5
Abstract
We 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. The 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. Ultra-low frequency base-substitution mutations can be detected directly by using the SMRT DNA sequencer, and this technology provides a phenotype-independent mutation assay.Keywords
Funding Information
- KAKENHI (Grant code:23221006)
- Health and Labour Sciences Research Grants for Research on Development of New Drugs (Grant code:KHB1209 and H26-souyaku-ippan-008)
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