False Positives in Multiplex PCR-Based Next-Generation Sequencing Have Unique Signatures
False Positives in Multiplex PCR-Based Next-Generation Sequencing Have Unique Signatures
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DOI：
10.1016/j.jmoldx.2014.06.001
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发表时间：
2014-09-01
影响因子：
4.1
通讯作者：
Eshleman, James R.
中科院分区：
文献类型：
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作者：
McCall, Chad M.;Mosier, Stacy;Eshleman, James R.
下一代测序技术通过对人类癌症中的多个基因进行快速突变分析而显示出巨大的前景。最近，我们实施了基于多重PCR的Ion AmpliSeq Cancer Hotspot Panel（50个基因中>200个扩增子），以评估肺和结直肠腺癌中的EGFR、KRAS和BRAF。在10%的样本中，自动分析发现了EGFR中的新型G873 R替代突变。通过单独检查读数，我们在291个样品中的50个中发现了>5%的读数中的这种突变，并且还在18个另外的扩增子中发现了类似的事件。这些明显的突变仅存在于短读段和读段两端的10个碱基内。因此，我们假设这些是来自与非靶向扩增子的几乎互补序列混杂结合的组引物。在19例病例中，18例突变周围的序列与样本组中的引物结合位点匹配，因此可能与样本组引物相对应。此外，由于大多数引物没有显示出这种效果，我们证明了下一代测序可以用于更好地设计多重PCR引物，通过迭代消除违规引物，以最大限度地减少错误引发。我们的研究结果表明，需要仔细的序列分析，以避免假阳性突变，可以出现在多重PCR面板。AmpliSeq癌症面板是临床诊断的宝贵工具，可以帮助人们了解潜在的伪影。
Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus Likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.