Smith-Roe Stephanie L, Hobbs Cheryl A, Hull Victoria, Auman J Todd, Recio Leslie, Streicker Michael A, Rivas Miriam V, Pratt Gabriel A, Lo Fang Yin, Higgins Jacob E, Schmidt Elizabeth K, Williams Lindsey N, Nachmanson Daniela, Valentine Charles C, Salk Jesse J, Witt Kristine L
Division of Translational Toxicology, NIEHS, Research Triangle Park, NC.
Integrated Laboratory Systems, LLC (an Inotiv company), Research Triangle Park, NC.
bioRxiv. 2023 May 9:2023.05.08.539833. doi: 10.1101/2023.05.08.539833.
Duplex sequencing (DuplexSeq) is an error-corrected next-generation sequencing (ecNGS) method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors by comparing grouped strand sequencing reads. The resulting background of less than one artifactual mutation per 10 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DuplexSeq-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues, a considerable advancement compared to currently used gene mutation assays.
DuplexSeq is an ultra-accurate NGS technology that directly quantifies mutationsENU-dependent mutagenesis was detected 24 h post-exposure in proliferative tissuesMultiple tissues exhibited the canonical ENU mutation spectrum 7 d after exposureResults obtained with DuplexSeq were highly concordant between laboratoriesThe Rat-50 Mutagenesis Assay is promising for applications in genetic toxicology.
双链测序（DuplexSeq）是一种经过错误校正的下一代测序（ecNGS）方法，其中分子条形码通过信息学将PCR拷贝与其源DNA链联系起来，从而能够通过比较成组的链测序读数在计算上消除错误。每10个核苷酸产生少于一个人为突变的背景允许直接检测体细胞突变。TwinStrand Biosciences公司开发了一种基于DuplexSeq的诱变试验来对大鼠基因组进行采样，该试验可应用于遗传毒性测试。为了评估该试验用于诱变早期检测的效果，使用雄性Hsd:Sprague Dawley SD大鼠（每组3只）进行了一项时间进程研究，通过灌胃给予单剂量40 mg/kg的N-乙基-N-亚硝基脲（ENU），在暴露后3小时、24小时、7天和28天分析胃、骨髓、血液和肝脏组织中的突变频率（MF）和谱。在ENU暴露的大鼠中，早在暴露后24小时，胃（接触部位）和骨髓（高度增殖组织）以及在暴露后7天肝脏和血液中的MF就出现了显著增加。在暴露后7天，所有四个组织中都确定了ENU的典型突变特征。对来自不同组织和时间点的一部分样本进行的实验室间分析表明，MF和谱都具有显著的可重复性。这些结果表明，通过直接对从各种组织获得的DNA靶向区域进行测序，可以成功评估MF和谱，与目前使用的基因突变试验相比有了相当大的进步。
DuplexSeq是一种超精确的NGS技术，可直接定量突变
在增殖组织中，暴露后24小时检测到ENU依赖性诱变
暴露7天后，多个组织呈现典型的ENU突变谱
DuplexSeq获得的结果在各实验室之间高度一致
大鼠-50诱变试验在遗传毒理学应用方面很有前景。
