Accurate detection of low frequency mutations from plasma cell-free DNA in blood using targeted next generation sequencing technology has shown promising benefits in clinical settings. Duplex sequencing technology is the most commonly used approach in liquid biopsies. Unique molecular identifiers are attached to each double-stranded DNA template, followed by production of low-error consensus sequences to detect low frequency variants. However, high sequencing costs have hindered application of this approach in clinical practice. Here, we have developed an improved duplex sequencing approach called SinoDuplex, which utilizes a pool of adapters containing pre-defined barcode sequences to generate far fewer barcode combinations than with random sequences, and implemented a novel computational analysis algorithm to generate duplex consensus sequences more precisely. SinoDuplex increased the output of duplex sequencing technology, making it more cost-effective. We evaluated our approach using reference standard samples and cell-free DNA samples from lung cancer patients. Our results showed that SinoDuplex has high sensitivity and specificity in detecting very low allele frequency mutations. The source code for SinoDuplex is freely available at https://github.com/SinOncology/sinoduplex.

使用靶向下一代测序技术从血液中无浆细胞的DNA准确检测低频突变已在临床环境中显示出令人鼓舞的益处。双工测序技术是液体活检中最常用的方法。独特的分子识别符附加到每个双链DNA模板上，然后产生低错误共有序列以检测低频变异。但是，高测序成本阻碍了该方法在临床实践中的应用。在这里，我们开发了一种改进的双工测序方法，称为SinoDuplex，该方法利用包含预定义条形码序列的衔接子池生成比随机序列少得多的条形码组合，并实现了新颖的计算分析算法以更精确地生成双链共有序列。SinoDuplex增加了双工测序技术的产出，使其更具成本效益。我们使用参考标准样品和肺癌患者的无细胞DNA样品评估了我们的方法。我们的结果表明，SinoDuplex在检测非常低的等位基因频率突变方面具有很高的灵敏度和特异性。SinoDuplex的源代码可从https：// github免费获得。