Mapping DNA polymerase errors by single-molecule sequencing
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CitationLee, David F., Jenny Lu, Seungwoo Chang, Joseph J. Loparo, and Xiaoliang S. Xie. 2016. “Mapping DNA polymerase errors by single-molecule sequencing.” Nucleic Acids Research 44 (13): e118. doi:10.1093/nar/gkw436. http://dx.doi.org/10.1093/nar/gkw436.
AbstractGenomic 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.
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