Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.
Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.
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DOI：
10.1093/nar/gkw224
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发表时间：
2016-06-20
影响因子：
14.9
通讯作者：
Godfrey TE
中科院分区：
文献类型：
--
作者：
Ståhlberg A;Krzyzanowski PM;Jackson JB;Egyud M;Stein L;Godfrey TE
液体活检中无细胞DNA的检测为非侵入性产前检测和作为癌症生物标记物提供了巨大的潜力。然而，这些样本中的胎儿和肿瘤DNA组分可能极低，需要超灵敏的方法来检测它们。在这里，我们报告了一种非常简单和快速的方法，将条形码引入到由5 ng DNA组成的DNA文库中。条形码接头引物设计具有寡核苷酸发夹结构，以在聚合酶链式反应(PCR)的第一轮中保护分子条形码，并防止它们参与错误启动事件。我们的方法支持高水平的多路复用和具有灵活的库内容的下一代测序库的构建。我们表明，可以生成1-、5-、13-和31-复合体的统一文库。利用条形码为每个原始DNA分子生成一致的读数，减少了背景测序噪音，并允许在克隆细胞系DNA和无细胞血浆DNA中检测频率低于0.1%的变异等位基因。因此，我们的方法弥合了数字PCR的高度敏感但特定的能力和下一代测序的广泛靶标能力之间的差距，前者只允许分析有限数量的变异，后者传统上缺乏检测罕见变异的敏感性。
Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.