Cytosine deamination is a major cause of baseline noise in next-generation sequencing.
Cytosine deamination is a major cause of baseline noise in next-generation sequencing.
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DOI：
10.1007/s40291-014-0115-2
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发表时间：
2014-10
影响因子：
4
通讯作者：
Eshleman, James R.
中科院分区：
文献类型：
--
作者：
Chen, Guoli;Mosier, Stacy;Gocke, Christopher D.;Lin, Ming-Tseh;Eshleman, James R.
随着下一代测序（NGS）成为临床诊断实验室中的主要测序平台，识别构成基线噪声并可能干扰低水平基因突变检测的伪影至关重要。这对于需要超灵敏检测的应用尤其重要，例如实体瘤的分子复发和癌症的早期检测。我们最近在野生型外周血和福尔马林固定石蜡包埋（FFPE）样本中观察到C：G > T：A突变比其他背景噪音高约10倍。我们假设这些可能代表使用其他平台观察到的胞嘧啶脱氨基作用事件。为了验证这一假设，我们用尿嘧啶N-糖基化酶（UNG）预处理样品。此外，我们还通过在不存在聚合酶的情况下使样品经受热循环来模拟与PCR热循环相关的热，测试了一些胞嘧啶脱氨基是否可能是实验室伪影。为了测试通用UNG预处理的安全性，我们测试了用UNG处理的已知阳性样本。UNG预处理显著减少了这些突变，与胞嘧啶脱氨基的生物来源一致。模拟热循环加热的样品显示出显著增加的C：G > T：A突变，而不影响其他基线碱基取代。具有已知突变的样本表明，在UNG治疗后，我们检测这些突变的能力没有降低。NGS期间的基线噪声主要是由于胞嘧啶脱氨基，其来源可能是生物和热循环的伪影，并且可以通过UNG预处理来减少。
As next generation sequencing (NGS) becomes a major sequencing platform in clinical diagnostic laboratories, it is critical to identify artifacts that constitute baseline noise and may interfere with detection of low level gene mutations. This is especially critical for applications requiring ultrasensitive detection, such as molecular relapse of solid tumors and early detection of cancer. We recently observed approximately ~10-fold higher C:G > T:A mutations than other background noise in both wild type peripheral blood and formalin fixed paraffin embedded (FFPE) samples. We hypothesized that these might represent cytosine deamination events that have been seen using other platforms. To test the hypothesis, we pretreated samples with uracil N-glycosylase (UNG). Additionally, we also tested whether some of the cytosine deamination might be laboratory artifact by simulating the heat associated with PCR thermocyling by subjecting samples to thermocycling in the absence of polymerase. To test the safety of universal UNG pretreatment, we tested known positive samples treated with UNG. UNG pretreatment significantly reduced these mutations, consistent with a biologic source for the cytosine deamination. The simulated thermocycling heated samples demonstrated significantly increased C:G > T:A mutations without affecting other baseline base substitutions. Samples with known mutations demonstrated no decrease in our ability to detect these after treatment with UNG. Baseline noise during NGS is mostly due to cytosine deamination, the source of which is likely both biologic and an artifact of thermocycling, and it can be reduced by UNG pretreatment.
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影响因子：
17.1
作者：
Leary RJ;Kinde I;Diehl F;Schmidt K;Clouser C;Duncan C;Antipova A;Lee C;McKernan K;De La Vega FM;Kinzler KW;Vogelstein B;Diaz LA Jr;Velculescu VE
通讯作者：
Velculescu VE
影响因子：
11.2
作者：
Heitzer, Ellen;Auer, Martina;Speicher, Michael R.
通讯作者：
Speicher, Michael R.
影响因子：
24.5
作者：
Kanda, Mitsuro;Knight, Spencer;Goggins, Michael
通讯作者：
Goggins, Michael
影响因子：
4.1
作者：
Lin, Ming-Tseh;Tseng, Li-Hui;Gocke, Christopher D.
通讯作者：
Gocke, Christopher D.
影响因子：
4.8
作者：
Sandigursky, M;Franklin, WA
通讯作者：
Franklin, WA