Error-corrected Duplex Sequencing enables direct detection and quantification of mutations in human TK6 cells with remarkable inter-laboratory consistency

Error-corrected Duplex Sequencing (DuplexSeq) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DuplexSeq to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two laboratories, TK6 cells were exposed to ENU (25-200 uM) and their DNA was sequenced 48, 72, and 96 h post-exposure. A DuplexSeq mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A robust increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (R2=0.95). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DuplexSeq for characterizing chemical mutagenicity in both research and regulatory evaluation.

纠错型双链测序（Error-corrected Duplex Sequencing，DuplexSeq）可实现低频突变的直接定量，在化学致突变性评估领域拥有巨大应用潜力。本研究探究了DuplexSeq在暴露于典型DNA烷化剂N-乙基-N-亚硝基脲（N-ethyl-N-nitrosourea，ENU）的人淋巴母细胞TK6中，定量诱导突变频率（mutation frequency，MF）及其突变谱的应用潜力。此外，本研究还探索了该应用场景下的适宜实验参数，并评估了实验室间的重现性。两家实验室分别将TK6细胞暴露于浓度为25~200 μM的ENU中，并于暴露后48、72、96小时提取细胞DNA进行测序。本研究采用靶向覆盖基因组内20个2.4 kb区域的DuplexSeq致突变性靶向测序panel，以采集具有基因组代表性的多样化序列背景。两家实验室均观测到MF显著升高，且该升高趋势不受采样时间的影响。两家实验室测得的MF浓度-反应曲线呈现极强的正相关关系（R²=0.95）。C:G>T:A、T:A>C:G、T:A>A:T及T:A>G:C四类突变在两家实验室中均呈现一致的浓度依赖性升高趋势，且所有采样时间点下C:G>T:A突变的占比均较高。不同靶位点在两家实验室中的响应模式相似，且基因间区域的平均MF水平更高。上述结果证实，DuplexSeq在突变频率与突变谱的检测中，于不同采样时间及实验室间均表现出极佳的重现性，表明其在化学致突变性的研究与监管评价中具备极高的应用价值。