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PacBio sequencing detects genome‐wide ultra‐low‐frequency substitution mutations resulting from exposure to chemical mutagens

Genetic toxicology uses several assays to identity mutagens and protects the public. Most of these assays, however, rely on reporter genes, can only measure mutation indirectly based on phenotype, and often require specific cell lines or animal models—features that impede their integration with existing and emerging toxicological models, such as organoids. In this study, we show that PacBio Single‐Molecule, Real‐Time (PB SMRT) sequencing identified substitution mutations caused by chemical mutagens in Escherichia coli by generating nearly error‐free consensus reads after repeatedly inspecting both strands of circular DNA molecules. Using DNA from E. coli exposed to ethyl methanosulfonate (EMS) or N‐ethyl‐N‐nitrosourea (ENU), PB SMRT sequencing detected mutation frequencies (MFs) and spectra comparable to those obtained by clone‐sequencing from the same exposures. The optimized background MF of PB SMRT sequencing was ≤ 1 × 10−7 mutations per base pair (mut/bp).