Mapping DNA polymerase errors by single-molecule sequencing.
Mapping DNA polymerase errors by single-molecule sequencing.
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DOI：
10.1093/nar/gkw436
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发表时间：
2016-07-27
影响因子：
14.9
通讯作者：
Xie XS
中科院分区：
文献类型：
--
作者：
Lee DF;Lu J;Chang S;Loparo JJ;Xie XS
DNA 聚合酶复制错误会损害基因组完整性，这种复制错误在整个基因组中以序列依赖性方式发生。准确、完整地量化 DNA 聚合酶的错误谱具有挑战性，因为错误很少且难以检测。我们报告了一种高通量测序分析，可在单分子水平上绘制体外 DNA 复制错误图谱。与以前的方法不同，我们的测定能够在任何模板底物上以碱基分辨率快速检测大量聚合酶错误，而没有定量偏差。为了克服高通量测序的高错误率，我们的测定使用条形码策略，其中每个复制产物在扩增前用独特的核苷酸序列标记。这样可以比较同一产品的多个测序读数，以便发现并消除测序错误。我们证明了我们的测定能够表征几种 DNA 聚合酶的平均错误率、错误热点和病变旁路保真度。
Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replication product is tagged with a unique nucleotide sequence before amplification. This allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases.