Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments
Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments
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DOI：
10.1186/s13059-014-0420-4
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发表时间：
2014-01-01
期刊：
影响因子：
12.3
通讯作者：
Frazer, Kelly A.
中科院分区：
文献类型：
--
作者：
Smith, Erin N.;Jepsen, Kristen;Frazer, Kelly A.
准确的等位基因频率对于测量亚克隆的异质性和克隆进化很重要。深度靶向测序数据可能包含重复的聚合酶链式反应，从而夸大了感知到的阅读深度。在这里，我们修改了Illumina TruSeq定制扩增试剂盒，使其包括单分子标签(SMT)，并表明SMT识别的重复项来自于PCR。我们证明，当不存在时，保留PCR重复读取可以意味着克隆进化，而它们的移除有效地控制了假阳性率。此外，聚合酶链式反应重复改变了对肿瘤样本中亚克隆异质性的估计。我们的方法简化了PCR重复鉴定，并在肿瘤异质性和克隆进化的研究中强调了它们的去除。
Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution.