Next-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency. Allele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%. For properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

中文翻译：

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一种简单的方法来估计下一代测序全外显子组测序分析中等位基因频率低的基因突变的内部检测限

下一代测序 (NGS) 深刻地改变了遗传/基因组研究的方法。特别是，NGS 在检测与疾病风险相关的突变方面的临床应用有助于开发有效的治疗策略。最近，通过 NGS 对体细胞基因突变的综合分析也被用作控制制造生物制药的细胞基质质量的新方法。然而，NGS 对细胞底物的质量评估很大程度上取决于罕见体细胞突变的检测限 (LOD)。本研究的目的是开发一种简单的方法来评估全外显子组测序 (WES) 通过 NGS 检测低等位基因频率突变的能力。为了估计低频体细胞突变的 WES 的 LOD，我们使用相同的 NGS 平台和分析设计反复独立地执行了参考基因组 DNA 的 WES。LOD 定义为相对标准偏差 (RSD) 值为 30% 的等位基因频率，并通过 RSD 与等位基因频率之间关系的移动平均曲线估计。参考材料中已通过液滴数字 PCR (ddPCR) 预验证的 20 个突变的等位基因频率从每次运行的 5、15、30 或 40 G 碱基对 (Gbp) 测序数据中获得。WES 测量的等位基因频率与 ddPCR 预验证的等位基因频率之间存在显着关联，其 p 值随着测序数据大小的增加而降低。通过这种方法，在测序数据为 15 Gbp 或以上的 WES 中，等位基因频率的 LOD 估计在 5% 到 10% 之间。为了正确解释体细胞基因突变的 WES 数据，需要有一个低等位基因频率的截止阈值。通过本研究中所示的简单方法估计的内部 LOD 为设置截止值提供了依据。