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

Area of Science:

• Genomics
• Toxicology
• Molecular Biology

Background:

• Error-corrected next-generation sequencing (ecNGS) is crucial for detecting rare mutations.
• Clonal and sub-clonal variants (CVs and SCVs) can reduce mutation detection sensitivity in sequencing studies.
• The TK6 human lymphoblastoid cell line is a standard model for genotoxicity testing.

Purpose of the Study:

• To apply Hawk-Seq™ for evaluating chemical-induced mutations in TK6 cells.
• To identify and filter out clonal and sub-clonal variants to improve mutation detection sensitivity.
• To establish a reliable method for assessing mutagenicity of chemicals like MNU and ENU.

Main Methods:

• Resequencing of the TK6 genome to identify 4,501,430 clonal variants (CVs).
• Filtering of common base substitutions (BSs) across vehicle controls to identify and remove sub-clonal variants (SCVs).
• Application of Hawk-Seq™ to TK6 cells treated with N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) after rigorous background filtering.

Main Results:

• Initial BS frequency in vehicle controls was 2.0 × 10^-6 bp, reduced to 0.65 × 10^-6 bp after filtering CVs and SCVs.
• MNU treatment resulted in a BS frequency of 9.0 × 10^-6 bp, predominantly G:C > A:T mutations.
• ENU treatment showed a BS frequency of 2.0 × 10^-6 bp, with increased G:C > A:T, A:T > C:G, and A:T > G:C mutations.

Conclusions:

• The developed method using Hawk-Seq™ with CV/SCV filtering significantly enhances the signal-to-noise ratio for mutation detection.
• The method accurately identified distinct mutagenic profiles for MNU and ENU, reflecting their known mechanisms.
• This approach holds potential for sensitive detection of mutations and elucidation of diverse mutagenic mechanisms in toxicological studies.