Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.
Institute of Bioinformatics, University Medicine Greifswald, Greifswald, Germany.
Health Phys. 2020 Jul;119(1):109-117. doi: 10.1097/HP.0000000000001251.
Little is known about the mutational impact of ionizing radiation (IR) exposure on a genome-wide level in mammalian tissues. Recent advancements in sequencing technology have provided powerful tools to perform exome-wide analyses of genetic variation. This also opened up new avenues for studying and characterizing global genomic IR-induced effects. However, genotypes generated by next generation sequencing (NGS) studies can contain errors, which may significantly impact the power to detect signals in common and rare variant analyses. These genotyping errors are not explicitly detected by the standard Genotype Analysis ToolKit (GATK) and Variant Quality Score Recalibration (VQSR) tool and thus remain a potential source of false-positive variants in whole exome sequencing (WES) datasets. In this context, the transition-transversion ratio (Ti/Tv) is commonly used as an additional quality check. In case of IR experiments, this is problematic when Ti/Tv itself might be influenced by IR treatment. It was the aim of this study to determine a suitable threshold for variant filters for NGS datasets from irradiated cells in order to achieve high data quality using Ti/Tv, while at the same time being able to investigate radiation-specific effects on the Ti/Tv ratio for different radiation doses. By testing a variety of filter settings and comparing the obtained results with publicly available datasets, we observe that a coverage filter setting of depth (DP) 3 and genotype quality (GQ) 20 is sufficient for high quality single nucleotide variants (SNVs) calling in an analysis combining GATK and VSQR and that Ti/Tv values are a consistent and useful indicator for data quality assessment for all tested NGS platforms. Furthermore, we report a reduction in Ti/Tv in IR-induced mutations in primary human gingiva fibroblasts (HGFs), which points to an elevated proportion of transversions among IR-induced SNVs and thus might imply that mismatch repair (MMR) plays a role in the cellular damage response to IR-induced DNA lesions.
关于电离辐射（IR）暴露对哺乳动物组织全基因组水平的突变影响知之甚少。测序技术的最新进展为进行外显子范围的遗传变异分析提供了强大的工具。这也为研究和描述全球基因组 IR 诱导效应开辟了新途径。然而，下一代测序（NGS）研究产生的基因型可能包含错误，这可能会显著影响在常见和罕见变体分析中检测信号的能力。这些基因分型错误没有被标准的基因型分析工具包（GATK）和变异质量评分重新校准（VQSR）工具明确检测到，因此仍然是全外显子测序（WES）数据集假阳性变体的潜在来源。在这种情况下，转换-颠换比（Ti/Tv）通常用作额外的质量检查。在 IR 实验中，当 Ti/Tv 本身可能受到 IR 处理的影响时，这是有问题的。本研究的目的是确定用于辐照细胞的 NGS 数据集的变体过滤器的合适阈值，以便使用 Ti/Tv 实现高质量的数据，同时能够研究不同辐射剂量下辐射对 Ti/Tv 比值的特异性影响。通过测试各种过滤器设置并将获得的结果与公开可用的数据集进行比较，我们观察到在结合 GATK 和 VSQR 进行的分析中，深度（DP）为 3 和基因型质量（GQ）为 20 的覆盖过滤器设置足以进行高质量的单核苷酸变体（SNV）调用，并且 Ti/Tv 值是所有测试 NGS 平台数据质量评估的一致且有用的指标。此外，我们报告在原发性人牙龈成纤维细胞（HGF）的 IR 诱导突变中 Ti/Tv 值降低，这表明 IR 诱导的 SNVs 中转换的比例升高，因此可能意味着错配修复（MMR）在细胞对 IR 诱导的 DNA 损伤的损伤反应中起作用。
