Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah, United States of America.
ARUP Laboratories, Salt Lake City, Utah, United States of America.
PLoS One. 2020 Feb 21;15(2):e0229063. doi: 10.1371/journal.pone.0229063. eCollection 2020.
Challenges with distinguishing circulating tumor DNA (ctDNA) from next-generation sequencing (NGS) artifacts limits variant searches to established solid tumor mutations. Here we show early and random PCR errors are a principal source of NGS noise that persist despite duplex molecular barcoding, removal of artifacts due to clonal hematopoiesis of indeterminate potential, and suppression of patterned errors. We also demonstrate sample duplicates are necessary to eliminate the stochastic noise associated with NGS. Integration of sample duplicates into NGS analytics may broaden ctDNA applications by removing NGS-related errors that confound identification of true very low frequency variants during searches for ctDNA without a priori knowledge of specific mutations to target.
从下一代测序（NGS）伪影中区分循环肿瘤 DNA（ctDNA）的挑战限制了变体搜索仅限于已建立的实体瘤突变。在这里，我们表明早期和随机 PCR 错误是 NGS 噪声的主要来源，尽管采用了双链分子条形码、去除由于不确定潜能克隆性造血引起的伪影以及抑制模式错误，这些噪声仍然存在。我们还证明了需要重复样本以消除与 NGS 相关的随机噪声。将重复样本整合到 NGS 分析中，可以通过消除与 NGS 相关的错误来扩大 ctDNA 的应用范围，这些错误在没有针对特定突变的先验知识的情况下，会混淆对 ctDNA 中真正极低频率变体的识别。
