A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens
A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens
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DOI：
10.1093/nar/gks610
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发表时间：
2012-08-01
影响因子：
14.9
通讯作者：
Tommasi, Stella
中科院分区：
文献类型：
--
作者：
Besaratinia, Ahmad;Li, Haiqing;Tommasi, Stella
许多致癌物在人类基因组中留下独特的突变指纹。这些突变指纹表现为特定类型的突变，通常聚集在暴露于致癌物的个体的肿瘤基因组中的某些基因组位点。为了开发检测致癌物突变指纹的高通量方法，我们制定了一种经济、时间和劳动力有效的策略，其中广泛使用的转基因Big Blue (R)小鼠突变检测分析与Roche/454 Genome Sequencer FLX Titanium下一代测序技术兼容。作为原理证明，我们使用这种新方法建立了三种具有不同诱变效力的主要致癌物的突变指纹，包括已知的强、中和弱诱变剂，包括阳光紫外线辐射、4-氨基联苯和二手烟。为了验证目的，我们将通过我们新开发的方法获得的这些致癌物的突变指纹与使用传统低通量方法平行分析获得的突变指纹进行了比较，即标准突变检测分析，然后使用毛细管DNA测序仪进行直接DNA测序。我们证明，这种基于高通量下一代测序的方法对于检测被测致癌物的突变指纹具有高度特异性和敏感性。该方法重现性好，准确度与现有的低通量方法相当。总之，这种新方法有可能通过对内源性和/或外源性基因毒性剂诱导的突变进行高通量分析来推动致癌领域的发展。
Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue (R) mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.