Genome-wide mutation analysis induced by mutagens in TK6 cells using Hawk-Seq™

Error-corrected next-generation sequencing (ecNGS) sensitively detects rare mutations in biological models. We applied Hawk-Seq™ to evaluate chemical-induced mutations using the IVGT TK6 human lymphoblastoid cell line. Since clonal and sub-clonal variants (CVs and SCVs) decrease mutation detection sensitivity, we first identified 4,501,430 CVs compared to GRCh38 by resequencing the TK6 genome. The overall base substitution (BS) frequency in vehicle controls after filtering out these variants was 2.0 × 10^-6 base pairs (bp), relatively higher than in other ecNGS studies. A total of 4974 sites provided the same types of BSs in ≥ 2 vehicle controls, suggesting that SCVs increased the error frequency. After filtering out these sites, the overall background BS frequency significantly decreased (0.93 × 10^-6 bp). Therefore, we filtered out the potential SCV positions identified using resequencing data with increased depth (mean depth of ca. 110), reducing the background overall BS frequency to 0.65 × 10^-6 bp. Finally, we evaluated DNA samples from TK6 cells exposed to N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) for 24 h. The overall BS frequencies in MNU- and ENU-treated samples were 9.0 × 10^-6 and 2.0 × 10^-6 bp, respectively, significantly improving the signal-to-noise ratio. MNU predominantly induced G:C > A:T (21 × 10^-6 bp), 62 times higher than that induced by vehicle controls. ENU primarily induced G:C > A:T (2.7 × 10^-6 bp) and significantly increased A:T > C:G and A:T > G:C frequencies (∼10^-7 bp). Our method sensitively detected mutations, including minor patterns, indicating its potential to reflect various mutagenic mechanisms.