Yang Zhiliang, Sun Guilian
Department of Pediatrics, the First Hospital of China Medical University, Shenyang, 110001, China.
Sci Rep. 2017 Oct 23;7(1):13751. doi: 10.1038/s41598-017-13116-6.
The GS Junior sequencer provides simplified procedures for library preparation and data processing. Errors in pyrosequencing generate some biases during library construction and emulsion PCR amplification. False-positive mutations are identified by related characteristics described in the manufacturer's manual, and some detected mutations may have 'borderline' characteristics when they are detected in few reads or at low frequency. Among these mutations, however, some may be true positives. This study aimed to improve the accuracy of identifying true positives among mutations with borderline false-positive characteristics detected with GS Junior sequencing. Mutations with the borderline features were tested for validity with Sanger sequencing. We examined 10 mutations detected in coverages <20-fold at frequencies >30% (group A) and 16 mutations detected in coverages >20-fold at frequencies < 30% (group B). In group A, two mutations were not confirmed, and two mutations with 100% frequency were confirmed as heterozygous alleles. No mutation in group B was confirmed. The two groups had significantly different false-positive prevalences (p = 0.001). These results suggest that mutations detected at frequencies less than 30% can be confidently identified as false-positives but that mutations detected at frequencies over 30%, despite coverages less than 20-fold, should be verified with Sanger sequencing.
GS Junior测序仪为文库制备和数据处理提供了简化程序。焦磷酸测序中的错误在文库构建和乳液PCR扩增过程中会产生一些偏差。通过制造商手册中描述的相关特征来识别假阳性突变，并且当一些检测到的突变在少数读数中或低频下被检测到时，可能具有“临界”特征。然而，在这些突变中，有些可能是真阳性。本研究旨在提高在GS Junior测序检测到的具有临界假阳性特征的突变中识别真阳性的准确性。用Sanger测序对具有临界特征的突变进行有效性测试。我们检测了在覆盖率<20倍且频率>30%时检测到的10个突变（A组）以及在覆盖率>20倍且频率<30%时检测到的16个突变（B组）。在A组中，两个突变未得到证实，两个频率为100%的突变被确认为杂合等位基因。B组未证实有突变。两组的假阳性发生率有显著差异（p = 0.001）。这些结果表明，频率低于30%检测到的突变可以确定为假阳性，但频率超过30%检测到的突变，尽管覆盖率低于20倍，仍应用Sanger测序进行验证。
