title: Mutation assay using single-molecule real-time (SMRT(TM)) sequencing technology.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Research Center for Environmental Quality Management, Kyoto University, Shiga, Japan ; Tomonari Matsuda, Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, Shiga 520-0811 Japan.
authors.forename: Tomonari
authors.initials: T
authors.lastname: Matsuda
authors.affiliation: Research Center for Environmental Quality Management, Kyoto University, Shiga, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Matsuda
authors.affiliation: National institute of Health Sciences, Tokyo, Japan.
authors.forename: Masami
authors.initials: M
authors.lastname: Yamada
keywords: Mutation assay; PacBio RSII DNA sequencer; Single-molecule real-time (SMRT) sequencing technology