CLAmp‐seq: A Novel Amplicon‐Based NGS Assay with Concatemer Error Correction for Improved Detection of Actionable Mutations in Plasma cfDNA from Patients with NSCLC
CLAmp‐seq: A Novel Amplicon‐Based NGS Assay with Concatemer Error Correction for Improved Detection of Actionable Mutations in Plasma cfDNA from Patients with NSCLC
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DOI：
10.1002/smtd.201900357
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发表时间：
2020-04
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通讯作者：
Lin Wang;Xiaomeng Hu;Qiaomei Guo;Xia Huang;Chia-hui Lin;X. Chen;Min Li;Qianqian Yao;Qianjun Zhou;Jianmin Wang;Shengrong Lin;Grace Q. Zhao;Li Weng;Kun Qian;J. Lou
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文献类型：
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作者：
Lin Wang;Xiaomeng Hu;Qiaomei Guo;Xia Huang;Chia-hui Lin;X. Chen;Min Li;Qianqian Yao;Qianjun Zhou;Jianmin Wang;Shengrong Lin;Grace Q. Zhao;Li Weng;Kun Qian;J. Lou
循环无细胞DNA（cfDNA）的高通量测序对于非小细胞肺癌（NSCLC）患者的靶向治疗选择和监测至关重要。然而，血液和测定伪影中较低量的cfDNA限制了下一代测序（NGS）测试的灵敏度和特异性。这项研究开发了一种新的基于扩增子的NGS技术，CLAmp‐seq（环连接扩增和测序），具有基于多联体的纠错策略，有效地消除了第一轮扩增中的聚合酶错误。分析验证显示，对于20 ng输入，突变等位基因频率（MAF）为0.1%时，中位检出率为100%。对于0.1%MAF，该测定分别实现了99.8%和99.5%的重复性和再现性一致性，并且对于0.5%MAF cfDNA标准品，两者均为100%。对134份NSCLC患者血浆样本的比较分析表明，CLAmp-seq NGS读数与液滴数字PCR（ddPCR）结果之间具有高度一致性（97.4%）和线性（R2= 0.95）。从预处理血浆样品获得的cfDNA中突变体的检测显示与组织基因分型结果的一致性为94.8%。在非癌性对照样品中未检测到突变。这表明CLAmp-seq NGS检测可以提供一种易于使用，快速，准确的分子诊断工具，具有多重能力，非常适合临床体外诊断。
High‐throughput sequencing of circulating cell‐free DNA (cfDNA) is critical for targeted therapy selection and monitoring in non‐small cell lung cancer (NSCLC) patients. However, low quantities of cfDNA in the blood and assay artifacts limit the sensitivity and specificity of next‐generation sequencing (NGS) test. This study develops a novel amplicon‐based NGS technology, CLAmp‐seq (circularligationamplification and sequencing), with a concatemer‐based error correction strategy, which effectively removes polymerase errors from the first round of amplification. Analytical validation shows median detection rate of 100% at a mutant allele frequency (MAF) of 0.1% for 20 ng of input. The assay achieves 99.8% and 99.5% concordance of repeatability and reproducibility for 0.1% MAF, respectively, and both 100% for 0.5% MAF cfDNA standards. A comparative analysis of 134 NSCLC patient plasma samples demonstrates strong concordance (97.4%) and linearity (R2= 0.95) between the CLAmp‐seq NGS readouts and droplet digital PCR (ddPCR) results. The detection of mutants in cfDNA obtained from pretreatment plasma samples shows 94.8% concordance with tissue genotyping results. No mutation is detected in noncancerous control samples. It shows that CLAmp‐seq NGS assay can offer an easy‐to‐use, fast, and accurate molecular diagnostic tool with multiplex capacity that is well‐suited for clinical in vitro diagnosis.