Background Mutation Frequencies in TK6 and L5178Y Cells: Implications for Error-Corrected Sequencing

Several error-corrected sequencing (ECS) methods can detect ultralow-frequency mutations and support mutagenicity assessments. While ECS can be applied to any DNA-containing sample, spontaneous mutations that accumulate in immortalized cell cultures-likely due to DNA replication errors-may elevate background mutation frequencies (MFs) and potentially confound ECS-based mutagenicity assessments. This study identified mutations unique to individual cells in TK6 and L5178Y populations by comparing the genomes of single-cell-derived clones to their parental cultures. These mutations resulted in MFs of 9 × 10^-7 and 6 × 10^-7 mutations per base pair (mut/bp) in commercially available TK6 and L5178Y cell populations, respectively. Freshly derived clonal populations from single TK6 and L5178Y cells exhibited lower MFs (0.5 × 10^-7 and 1 × 10^-7 mut/bp, respectively). These results suggest that commercially available TK6 and L5178Y cell populations have accumulated significant levels of background mutations that could affect the interpretation of ECS experiments. To test this hypothesis, commercially available and freshly derived clonal TK6 cell populations were grown for 5 days in medium containing 0.5, 2, and 8 μg/mL of the in vitro mutagen N4-hydroxycytidine and analyzed by HiFi sequencing, an ECS method. The results showed that freshly derived clonal populations had lower background MFs and greater relative MF fold increases upon mutagen exposure than the commercially available cell population. An alternative data analysis approach, based on absolute MF changes within each cell population, yielded more comparable results for commercial and clonal populations. These findings underscore the impact of background MFs on in vitro ECS analyses.