Background: Cell-free DNA (cfDNA) promises to serve as surrogate biomarkers for non-invasive molecular diagnostics. Disease-specific cfDNA, such as circulating tumor DNA (ctDNA), was short and rare, making the detection performance of the current targeted sequencing methods unsatisfying. Methods: Through introducing a linear pre-amplification process and optimizing the adapter ligation with customized reagents, we developed the One-PrimER Amplification (OPERA) system. In this study, we examined its performance in detecting mutations of low variant allelic frequency (VAF) in various samples with short-sized DNA fragments. Results: In cell line-derived samples containing sonication-sheared DNA fragments with 50-150 bp (peak at 70-80 bp), OPERA was capable of detecting mutations as low as 0.0025% VAF, while CAPP-Seq only detected mutations of >0.03% VAF. Both single nucleotide variant and insertion/deletion can be detected by OPERA. In synthetic fragments as short as 80 bp with low VAF (0.03%-0.1%), the detection sensitivity of OPERA was significantly higher compared to that of droplet digital polymerase chain reaction. The error rate was 5.9x10-5 errors per base after de-duplication in plasma samples collected from healthy volunteers. By suppressing single-strand errors, the error rate can be further lowered by >5 folds in EGFR T790M hotspot. In plasma samples collected from lung cancer patients, OPERA detected mutations in 57.1% stage I patients with 100% specificity and achieved a sensitivity of 30.0% in patients with tumor volume of less than 1 cm3. Conclusions: OPERA can effectively detect mutations in rare and highly-fragmented DNA. Trial registration: This study has been registered on ChiCTR (ChiCTR1900024028) at 23rd June 2019.

中文翻译：

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一种检测短而稀有无细胞 DNA 突变的超灵敏方法

背景：游离 DNA (cfDNA) 有望作为非侵入性分子诊断的替代生物标志物。疾病特异性 cfDNA，如循环肿瘤 DNA (ctDNA)，短且稀少，使得当前靶向测序方法的检测性能不尽如人意。方法：通过引入线性预扩增过程和使用定制试剂优化接头连接，我们开发了 One-Primer Amplification (OPERA) 系统。在这项研究中，我们检查了它在检测具有短 DNA 片段的各种样本中的低变异等位基因频率 (VAF) 突变方面的性能。结果：在含有 50-150 bp（峰值为 70-80 bp）的超声剪切 DNA 片段的细胞系衍生样本中，OPERA 能够检测低至 0.0025% VAF 的突变，而 CAPP-Seq 仅检测 > 0。03% VAF。OPERA 可以检测单核苷酸变异和插入/缺失。在低 VAF (0.03%-0.1%) 的短至 80 bp 的合成片段中，与液滴数字聚合酶链反应相比，OPERA 的检测灵敏度明显更高。对从健康志愿者收集的血浆样本进行去重后，错误率为每个碱基 5.9x10-5 个错误。通过抑制单链错误，EGFR T790M 热点中的错误率可以进一步降低 > 5 倍。在从肺癌患者收集的血浆样本中，OPERA 在 57.1% 的 I 期患者中检测到突变，特异性为 100%，并且在肿瘤体积小于 1 立方厘米的患者中达到 30.0% 的灵敏度。结论：OPERA 可以有效地检测稀有和高度片段化的 DNA 中的突变。试用注册：