“去重”在肺癌NGS数据分析中的重要作用

目的 探讨“不去重”和“去重”两种方法分析后各NGS相关指标间的差异，研究“去重”在靶向捕获NGS数据分析中的重要作用。方法 通过对58例肺癌患者的DNA样本进行靶向286基因探针杂交方法建库并进行NGS检测，每例NGS检测数据分别进行“去重”和“不去重”两种方法的生物信息学分析，比较两种方法分析得到的NGS相关指标Mapped Reads（可比对上reads比例）、On Target（靶向区域reads比例）、Mean Depth（平均测序深度）以及Uniformity（均一性）等数据之间的差异。结果 “不去重”和“去重”两种方法分析得到平均Mapped Reads、On Target、Mean Depth和Uniformity值，各组指标间差异均有统计学意义（P<0.001）。“去重”方法分析时，Mapped Reads、On Target和MeanDepth 3个指标均呈现出血浆样本与其他3种类型样本间（FFPE、穿刺和手术）差异有统计学意义，在Uniformity指标上则呈现出4种类型样本间差异均无统计学意义。而“不去重”方法分析后结果正好相反。结论 去除PCR扩增所致重复序列的“去重”步骤在NGS数据分析中具有重要的作用，能够改善结果均一性，真实反映所测样本的DNA模板数量及等位基因频率（AF值，allele frequency）。“去重”后结果更有助于临床决策。

Important Role of Deduplication Method in Next Generation Sequencing Data Analysis of Non-small Cell Lung Cancer

Objective To investigate the important role of deduplication method in next generation sequencing(NGS) data analysis by comparing the difference of indicators obtained from deduplication and duplication methods. Methods Tumor samples of a cohort of 58 NSCLC patients were collected. A panel of 286 genes was tested by NGS. The NGS data were analyzed by de-duplication method and common duplication method, respectively. Indicators of “Mapped Reads, On Target, Mean Depth and Uniformity” were compared. Results The differences of Mapped Reads, On Target, Mean Depth and Uniformity were statistically significant between two methods, respectively(P<0.001). Mapped Reads and On Target and Mean Depth analyzed by de-duplication method were found significantly different between plasma sample and other three types of samples, ie., formalin fixed and paraffin embedded(FFPE) sample and puncture biopsy and surgical tissue sample; while Uniformity was generated without significant difference between the four types of samples. The results by duplication analysis were opposite. Conclusion Deduplication step plays an important role in NGS data analysis, which could improve the Uniformity and reflect the real DNA template amount and allele frequency of genomic alterations. Deduplication result is helpful for clinical decision.