Single-tube, highly parallel mutation enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR.
Single-tube, highly parallel mutation enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR.
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DOI：
10.1373/clinchem.2014.228361
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发表时间：
2015-01
影响因子：
9.3
通讯作者：
Makrigiorgos MG
中科院分区：
文献类型：
--
作者：
Castellanos-Rizaldos E;Richardson K;Lin R;Wu G;Makrigiorgos MG
低水平突变的多重检测对许多技术提出了技术挑战，包括用于靶向重测序的癌症基因组。在具有异质性的肿瘤、具有基质污染的样品或生物流体中低于约2-5%丰度的突变的分析是有问题的，因为来自测序错误的“噪声”增加。通过深度测序降低噪声的技术必然会降低通量并增加成本。在这里，我们提供了COLD-PCR技术能够在癌症基因组中进行多重低水平突变检测，同时保持通量的原理证明。我们开发了一种多重耐温COLD-PCR（快速TT-COLD-PCR）方法，该方法使用为大规模平行测序开发的癌症基因组。在从基因组DNA进行多重预扩增后，我们将尾部连接到所有扩增子并进行快速TT-COLD-PCR。该方法以逐步方式逐渐增加变性温度，使得涵盖所有可能的变性温度。通过引入修饰的核苷酸，快速COLD-PCR适于在单个管中富集所有扩增子上的Tm增加突变以及Tm减少突变。使用定制的和商业化的基因板，包含8，50，190或16，000个扩增子，我们证明了快速TT-COLD-PCR同时富集了所有检测靶标上的突变。掺入dITP/dDTP代替dGTP/dATP能够富集Tm增加突变。系列稀释实验证明，使用Ion-Torrent的检测限约为0.01-0.1%突变丰度，使用桑格测序的检测限为0.1-0.3%。Fast-TT-COLD-PCR通过在多重单管反应中实现突变富集，提高了癌症基因组的检测限。COLD-PCR的这种新适应将亚克隆突变转化为克隆，从而便于检测和随后的突变测序。
Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-re-sequencing. Analysis of mutations below ~2–5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids, is problematic due to increased ‘noise’ from sequencing errors. Technologies that reduce noise via deep-sequencing unavoidably reduce throughput and increase cost. Here we provide proof-of-principle that COLD-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput. We have developed a multiplex temperature-tolerant-COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. Following a multiplex pre-amplification from genomic DNA we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for Tm-increasing as well as Tm-decreasing mutations over all amplicons, in a single tube. Using custom-made and commercial gene panels containing 8, 50, 190 or 16,000 amplicons we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of dITP/dDTP in place of dGTP/dATP enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit-of-detection of ~ 0.01–0.1% mutation abundance using Ion-Torrent and 0.1–0.3% using Sanger sequencing. Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing.
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影响因子：
14.9
作者：
Flaherty P;Natsoulis G;Muralidharan O;Winters M;Buenrostro J;Bell J;Brown S;Holodniy M;Zhang N;Ji HP
通讯作者：
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影响因子：
9.3
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通讯作者：
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影响因子：
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影响因子：
9.3
作者：
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通讯作者：
Makrigiorgos GM