Data from: Benefits and challenges with applying unique molecular identifiers in next generation sequencing to detect low frequency mutations

Indexing individual template molecules with a unique identifier (UID) before PCR and deep sequencing is promising for detecting low frequency mutations, as true mutations could be distinguished from PCR errors or sequencing errors based on consensus among reads sharing same index. In an effort to develop a robust assay to detect from urine low-abundant bladder cancer cells carrying well-documented mutations, we have tested the idea first on a set of mock templates, with wild type and known mutants mixed at defined ratios. We have measured the combined error rate for PCR and Illumina sequencing at each nucleotide position of three exons, and demonstrated the power of a UID in distinguishing and correcting errors. In addition, we have demonstrated that PCR sampling bias, rather than PCR errors, challenges the UID-deep sequencing method in faithfully detecting low frequency mutation.在聚合酶链式反应（PCR）与深度测序前，为每个模板分子赋予唯一识别符（Unique Identifier, UID）进行索引，该策略在低频突变检测领域极具应用潜力。基于共享同一索引的测序读段间的序列一致性，可将真实突变与PCR误差、测序误差精准区分开来。为开发一种可从尿液中检测携带已有充分文献记载突变的低丰度膀胱癌细胞的可靠检测方法，我们首先在一系列模拟模板上验证了该思路：将野生型序列与已知突变体按确定比例混合。我们测定了三个外显子每个核苷酸位点上PCR与Illumina测序的联合误差率，并验证了UID在区分与校正误差方面的效能。此外，我们还证实，相较于PCR误差，PCR采样偏差才是UID-深度测序方法在精准检测低频突变时面临的主要挑战。