Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing
Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing
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DOI：
10.1101/2020.06.28.176685
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发表时间：
2020-06
影响因子：
11.1
通讯作者：
C. Valentine;R. Young;M. Fielden;R. Kulkarni;L. Williams;Tan Li;Sheroy Minocherhomji;J. Salk
中科院分区：
文献类型：
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作者：
C. Valentine;R. Young;M. Fielden;R. Kulkarni;L. Williams;Tan Li;Sheroy Minocherhomji;J. Salk
错误校正下一代测序（ecNGS）可用于快速检测和量化任何组织、任何物种、任何基因组位置的环境暴露或内源性过程的体内诱变影响。与现有方法相比，ecNGS具有更快的速度、更高的可扩展性、更丰富的数据输出以及跨物种和跨位点的适用性，使其成为突变研究、监管安全性测试和新兴临床应用的强大新工具。准确测量突变的能力对于基础研究和识别潜在的药物和化学致癌物至关重要。目前体内定量诱变的方法是有限的，因为它们依赖于转基因啮齿动物系统，这种系统低通量、昂贵、耗时，并且不能完全代表其他物种，如人类。下一代测序（NGS）在概念上是检测任何生物体DNA突变的一个有吸引力的替代方案；然而，标准NGS的分辨率限制很差。NGS的技术错误率（~ 1 × 10−3）掩盖了体细胞突变的真实丰度，体细胞突变可以存在于每核苷酸频率≤1 × 10−7。利用双工测序技术，一种非常精确的错误校正NGS （ecNGS）技术，我们能够在暴露后31天内检测到两种小鼠品系的五个组织中三种致癌物引起的突变。我们观察到双工测序测量的突变诱导与金标准转基因啮齿动物突变测定之间存在很强的相关性。我们通过碱基取代的三核苷酸模式确定了每种化合物的暴露特异性突变谱。我们观察到突变易感性随基因组区域以及DNA链的变化。我们还在一种易患癌症的小鼠品系中发现了一种致癌的原始标记物，在致癌物质暴露不到一个月后，携带激活致癌基因的细胞克隆扩增证明了这一点。这些发现表明，ecNGS是一种有效的方法，可以灵敏地检测和表征突变和癌变的早期克隆进化特征。双工测序可以广泛应用于基础突变研究、监管安全性测试和新兴临床应用。
Significance Error-corrected next-generation sequencing (ecNGS) can be used to rapidly detect and quantify the in vivo mutagenic impact of environmental exposures or endogenous processes in any tissue, from any species, at any genomic location. The greater speed, higher scalability, richer data outputs, and cross-species and cross-locus applicability of ecNGS compared to existing methods make it a powerful new tool for mutational research, regulatory safety testing, and emerging clinical applications. The ability to accurately measure mutations is critical for basic research and identifying potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and do not fully represent other species such as humans. Next-generation sequencing (NGS) is a conceptually attractive alternative for detecting mutations in the DNA of any organism; however, the limit of resolution for standard NGS is poor. Technical error rates (∼1 × 10−3) of NGS obscure the true abundance of somatic mutations, which can exist at per-nucleotide frequencies ≤1 × 10−7. Using duplex sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by three carcinogens in five tissues of two strains of mice within 31 d following exposure. We observed a strong correlation between mutation induction measured by duplex sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified a primordial marker of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex sequencing can be broadly applied to basic mutational research, regulatory safety testing, and emerging clinical applications.