COLD-PCR enrichment of rare cancer mutations prior to targeted amplicon resequencing.
COLD-PCR enrichment of rare cancer mutations prior to targeted amplicon resequencing.
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DOI：
10.1373/clinchem.2011.176198
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发表时间：
2012-03
影响因子：
9.3
通讯作者：
Makrigiorgos GM
中科院分区：
文献类型：
--
作者：
Milbury CA;Correll M;Quackenbush J;Rubio R;Makrigiorgos GM
尽管人们对下一代测序（NGS）应用于个体癌症标本的突变分析产生了广泛的兴趣，但部分由于技术障碍，个性化临床基因组学的启动目前陷入停滞。由于肿瘤是遗传异质性的，并且经常与正常/基质细胞混合，因此产生的低丰度DNA体细胞突变经常产生模糊的结果或低于当前的NGS检测限，从而阻碍符合临床灵敏度/特异性标准的突变识别。在这里，我们研究了应用COLD-PCR的可行性，COLD-PCR是一种选择性放大突变的PCR形式，在将基于NGS的扩增子重新测序应用于临床样本之前，可以提高未知罕见体细胞突变的检测。我们使用COLD-PCR或常规PCR将来自连续稀释的含有突变的人类细胞系的DNA扩增为野生型（WT）DNA，以及肺腺癌和结直肠癌标本以进行比较。在TP 53、KRAS、IDH 1和EGFR区域的单独扩增后，将PCR产物条形码化，合并用于文库制备，并在Illumina-HiSeq 2000平台上测序。无论测序深度如何，测序错误决定了NGS分析的常规PCR扩增子中约1-2%突变丰度的突变检测限。相比之下，COLD-PCR扩增子使真正的突变超过序列噪声水平，从而允许可靠地鉴定约0.04%的突变丰度。测序深度不是鉴定COLD-PCR放大突变的重要因素。分析的临床标本显示了几个TP 53和KRAS错义突变，这些突变在常规扩增子的NGS后不能被称为，但在COLD-PCR扩增子中可以清楚地检测到。在一些样本中发现TP 53基因的广泛的肿瘤异质性。随着癌症护理转向个性化干预，基于每个患者肿瘤基因组中独特的遗传异常，我们预计COLD-PCR-NGS将阐明肿瘤中罕见突变的作用，实现基于NGS的多样化临床标本分析以及NGS与临床实践的广泛相互作用。
Despite widespread interest in the application of next-generation-sequencing (NGS) to the mutation profiling of individual cancer specimens, the onset of personalized clinical genomics is currently stalled due in part to technical hurdles. As tumors are genetically-heterogeneous and often mixed with normal/stromal cells, the resulting low-abundance DNA somatic mutations often produce ambiguous results or fall below the current NGS detection limit, thus hindering mutation calling that abides to clinical sensitivity/specificity standards. Here we examine the feasibility of applying COLD-PCR, a form of PCR that magnifies selectively the mutations, to boost the detection of unknown rare somatic mutations prior to applying NGS-based amplicon re-sequencing to clinical samples. We amplified DNA from serially-diluted mutation-containing human cell-lines into wild-type (WT) DNA, as well as lung adenocarcinoma and colorectal cancer specimens using COLD-PCR or conventional PCR for comparison. Following individual amplification of TP53, KRAS, IDH1, and EGFR regions, PCR products were barcoded, pooled for library preparation and sequenced on the Illumina-HiSeq2000 platform. Regardless of sequencing depth, sequencing errors dictated a mutation-detection limit of ~1–2% mutation abundance in conventional PCR amplicons analyzed by NGS. In contrast, COLD-PCR amplicons enabled genuine mutations to exceed the sequence noise levels, thus allowing reliable identification of mutation abundances of ~0.04%. Sequencing depth was not a significant factor in the identification of COLD-PCR-magnified mutations. The analyzed clinical specimens revealed several TP53 and KRAS missense mutations that could not be called following NGS of conventional amplicons, yet were clearly detectable in COLD-PCR amplicons. Extensive tumor heterogeneity in the TP53 gene was revealed in some samples. As cancer care shifts toward personalized intervention, based on the unique genetic abnormalities in each patient’s tumor genome, we anticipate that COLD-PCR-NGS will elucidate the role of rare mutations in tumors, enable NGS-based analysis of diverse clinical specimens and the broad inter-phasing of NGS with clinical practice.
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影响因子：
3.9
作者：
Li, Jin;Milbury, Coren A.;Li, Cheng;Makrigiorgos, G. Mike
通讯作者：
Makrigiorgos, G. Mike
影响因子：
12.3
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Kelso J
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Hainaut, P
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Flanagan, Adrienne M.
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14.9
作者：
Casbon JA;Osborne RJ;Brenner S;Lichtenstein CP
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Lichtenstein CP