Division of Translational Toxicology, NIEHS, Research Triangle Park, NC, USA.
Integrated Laboratory Systems, LLC (An Inotiv Company), Research Triangle Park, NC, USA.
Mutat Res Genet Toxicol Environ Mutagen. 2023 Oct;891:503669. doi: 10.1016/j.mrgentox.2023.503669. Epub 2023 Aug 3.
Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. 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 DS-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 in vivo gene mutation assays.
双脱氧测序（DS）是一种经过纠错的下一代测序方法，其中分子条形码将 PCR 拷贝信息链接回其源 DNA 链，从而能够在共识序列中计算去除错误。每个核苷酸少于一个人为突变的背景允许直接检测体细胞突变。TwinStrand Biosciences, Inc. 开发了一种基于 DS 的诱变测定法来对大鼠基因组进行采样，该方法可应用于遗传毒性测试。为了评估该测定法在诱变早期检测中的应用，对雄性 Hsd:Sprague Dawley SD 大鼠（每组 3 只）进行了一项时间进程研究，它们通过灌胃接受了 40 mg/kg N-乙基-N-亚硝脲（ENU）的单次剂量，在接触后 3 h、24 h、7 d 和 28 d 时对胃、骨髓、血液和肝脏组织中的突变频率（MF）和谱进行分析。ENU 暴露的大鼠在接触后 24 h 时胃（接触部位）和骨髓（高度增殖组织）的 MF 就显著增加，而在 7 d 时肝脏和血液的 MF 也显著增加。ENU 的典型突变特征在接触后 7 d 在所有四个组织中都建立起来。对不同组织和时间点的部分样本进行的实验室间分析表明，MF 和谱都具有出色的重现性。这些结果表明，可以通过直接测序从各种组织中获得的靶向 DNA 区域成功评估 MF 和谱，与目前使用的体内基因突变测定法相比，这是一个重大进展。
