Mapping DNA polymerase errors by single-molecule sequencing

Lee, David F.; Lu, Jenny; Chang, Seungwoo; Loparo, Joseph J.; Xie, Xiaoliang S.

doi:10.1093/nar/gkw436

Title: Mapping DNA polymerase errors by single-molecule sequencing

Journal Article · Mon May 16 00:00:00 EDT 2016 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkw436· OSTI ID:1360141

^[1]; Lu, Jenny ^[1]; Chang, Seungwoo ^[2]; Loparo, Joseph J. ^[2]; Xie, Xiaoliang S. ^[1]

Harvard Univ., Cambridge, MA (United States)
Harvard Medical School, Boston, MA (United States)

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. Here, 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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Research Organization:: Harvard Univ., Cambridge, MA (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: SC0012411

OSTI ID:: 1360141

Journal Information:: Nucleic Acids Research, Vol. 44, Issue 13; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 20 works

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