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

Area of Science:

• Genomics and Molecular Biology
• Toxicology and Mutagenesis

Background:

• Error-corrected sequencing (ECS) methods are crucial for detecting ultralow-frequency mutations and assessing mutagenicity.
• Spontaneous mutations accumulating in immortalized cell cultures can elevate background mutation frequencies (MFs), potentially confounding ECS results.
• Commercially available TK6 and L5178Y cell populations exhibit higher background MFs compared to freshly derived clonal populations.

Purpose of the Study:

• To investigate the impact of background mutations in commercially available cell lines on ECS-based mutagenicity assessments.
• To compare the performance of commercially available cell populations versus freshly derived clonal populations in ECS experiments.
• To evaluate the influence of different data analysis approaches on interpreting ECS results in the presence of background mutations.

Main Methods:

• Genomic comparison of single-cell-derived clones with parental TK6 and L5178Y cell cultures to identify unique mutations.
• Culturing commercially available and freshly derived TK6 cell populations with varying concentrations of the in vitro mutagen N4-hydroxycytidine.
• Analysis of mutagenicity using HiFi sequencing, an ECS method, and application of both relative and absolute mutation frequency change analyses.

Main Results:

• Commercially available TK6 and L5178Y cell populations showed significantly higher background MFs (9 × 10⁻⁷ and 6 × 10⁻⁷ mut/bp) than freshly derived clones (0.5 × 10⁻⁷ and 1 × 10⁻⁷ mut/bp).
• Freshly derived clonal TK6 populations exhibited lower background MFs and greater relative fold increases in MFs upon mutagen exposure compared to commercial populations.
• An absolute MF change analysis approach provided more comparable results between commercial and clonal cell populations than relative fold increase analysis.

Conclusions:

• Elevated background MFs in commercially available cell lines can impact the interpretation of in vitro ECS mutagenicity experiments.
• Freshly derived clonal cell populations offer a more reliable baseline for ECS-based mutagenicity assessments due to lower background MFs.
• The choice of data analysis method (relative vs. absolute MF changes) can influence the comparability of results across different cell population types.