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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.

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