High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.
High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.
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DOI：
10.1093/dnares/dsv010
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发表时间：
2015-08
期刊：
影响因子：
--
通讯作者：
Kato K
中科院分区：
文献类型：
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作者：
Kukita Y;Matoba R;Uchida J;Hamakawa T;Doki Y;Imamura F;Kato K
循环肿瘤DNA（ctDNA）是癌症研究的一个新兴领域。然而，由于技术限制，目前的ctDNA分析通常限于一个或几个突变位点。在大规模并行DNA测序仪的情况下，由高读取错误率引起的假阳性数量是一个主要问题。此外，由于模板制备期间的全局扩增步骤，最终序列读数不代表原始DNA群体。我们建立了一个高保真的目标测序系统的个别分子鉴定血浆无细胞DNA使用条形码序列;该系统包括以下两个步骤。(i)一种通过接头连接添加条形码序列的新型靶测序方法。该方法使用线性扩增来消除在聚合酶链反应的早期循环期间引入的误差。(ii)监控和删除错误的条形码标签。该过程涉及鉴定已测序的单个分子，并且其突变数量已绝对定量。使用来自胃癌或肺癌患者的血浆无细胞DNA，我们证明该系统几乎完全消除了假阳性，并能够从头检测和绝对定量血浆无细胞DNA中的突变。
Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA.