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Title: Mapping DNA polymerase errors by single-molecule sequencing

Abstract

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.

Authors:
ORCiD logo [1];  [1];  [2];  [2];  [1]
+ Show Author Affiliations
  1. Harvard Univ., Cambridge, MA (United States)
  2. Harvard Medical School, Boston, MA (United States)
Publication Date:
Research Org.:
Harvard Univ., Cambridge, MA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1360141
Grant/Contract Number:  
SC0012411
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 44; Journal Issue: 13; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; dna; dna-directed dna polymerase; amplification; bar codes; molecule

Citation Formats

Lee, David F., Lu, Jenny, Chang, Seungwoo, Loparo, Joseph J., and Xie, Xiaoliang S. Mapping DNA polymerase errors by single-molecule sequencing. United States: N. p., 2016. Web. doi:10.1093/nar/gkw436.
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Lee, David F., Lu, Jenny, Chang, Seungwoo, Loparo, Joseph J., & Xie, Xiaoliang S. Mapping DNA polymerase errors by single-molecule sequencing. United States. https://doi.org/10.1093/nar/gkw436
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Lee, David F., Lu, Jenny, Chang, Seungwoo, Loparo, Joseph J., and Xie, Xiaoliang S. Mon . "Mapping DNA polymerase errors by single-molecule sequencing". United States. https://doi.org/10.1093/nar/gkw436. https://www.osti.gov/servlets/purl/1360141.
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@article{osti_1360141,
title = {Mapping DNA polymerase errors by single-molecule sequencing},
author = {Lee, David F. and Lu, Jenny and Chang, Seungwoo and Loparo, Joseph J. and Xie, Xiaoliang S.},
abstractNote = {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.},
doi = {10.1093/nar/gkw436},
journal = {Nucleic Acids Research},
number = 13,
volume = 44,
place = {United States},
year = {Mon May 16 00:00:00 EDT 2016},
month = {Mon May 16 00:00:00 EDT 2016}
}
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https://doi.org/10.1093/nar/gkw436
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Works referenced in this record:

High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase
journal, January 1990

High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase
journal, January 1990

A unique error signature for human DNA polymerase ν
journal, February 2007

Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase
journal, August 1988

  • Tindall, Kenneth R.; Kunkel, Thomas A.
  • Biochemistry, Vol. 27, Issue 16
  • DOI: 10.1021/bi00416a027

Measuring Cation Dependent DNA Polymerase Fidelity Landscapes by Deep Sequencing
journal, August 2012

A unique error signature for human DNA polymerase ν
journal, February 2007

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase
journal, January 1988

Detection and quantification of rare mutations with massively parallel sequencing
journal, May 2011

  • Kinde, I.; Wu, J.; Papadopoulos, N.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 23
  • DOI: 10.1073/pnas.1105422108

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase
journal, January 1988

Measuring Cation Dependent DNA Polymerase Fidelity Landscapes by Deep Sequencing
journal, August 2012

Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase
journal, August 1988

  • Tindall, Kenneth R.; Kunkel, Thomas A.
  • Biochemistry, Vol. 27, Issue 16
  • DOI: 10.1021/bi00416a027

Streisinger Revisited: DNA Synthesis Errors Mediated by Substrate Misalignments
journal, January 2000

  • Bebenek, K.; Kunkel, T. A.
  • Cold Spring Harbor Symposia on Quantitative Biology, Vol. 65, Issue 0
  • DOI: 10.1101/sqb.2000.65.81

Polymerase specific error rates and profiles identified by single molecule sequencing
journal, February 2016

  • Hestand, Matthew S.; Houdt, Jeroen Van; Cristofoli, Francesca
  • Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 784-785
  • DOI: 10.1016/j.mrfmmm.2016.01.003

Y-family DNA polymerases and their role in tolerance of cellular DNA damage
journal, February 2012

  • Sale, Julian E.; Lehmann, Alan R.; Woodgate, Roger
  • Nature Reviews Molecular Cell Biology, Vol. 13, Issue 3
  • DOI: 10.1038/nrm3289

DNA damage tolerance: when it's OK to make mistakes
journal, January 2009

  • Chang, Debbie J.; Cimprich, Karlene A.
  • Nature Chemical Biology, Vol. 5, Issue 2
  • DOI: 10.1038/nchembio.139

Fidelity of Escherichia coli DNA Polymerase IV : PREFERENTIAL GENERATION OF SMALL DELETION MUTATIONS BY dNTP-STABILIZED MISALIGNMENT
journal, July 2002

  • Kobayashi, Sawami; Valentine, Michael R.; Pham, Phuong
  • Journal of Biological Chemistry, Vol. 277, Issue 37
  • DOI: 10.1074/jbc.M204826200

DNA damage tolerance: when it's OK to make mistakes
journal, January 2009

  • Chang, Debbie J.; Cimprich, Karlene A.
  • Nature Chemical Biology, Vol. 5, Issue 2
  • DOI: 10.1038/nchembio.139

Polymerase specific error rates and profiles identified by single molecule sequencing
journal, February 2016

  • Hestand, Matthew S.; Houdt, Jeroen Van; Cristofoli, Francesca
  • Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 784-785
  • DOI: 10.1016/j.mrfmmm.2016.01.003

Y-family DNA polymerases and their role in tolerance of cellular DNA damage
journal, February 2012

  • Sale, Julian E.; Lehmann, Alan R.; Woodgate, Roger
  • Nature Reviews Molecular Cell Biology, Vol. 13, Issue 3
  • DOI: 10.1038/nrm3289

Sequence error storms and the landscape of mutations in cancer
journal, August 2012

  • Kirsch, Stefan; Klein, Christoph A.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 36
  • DOI: 10.1073/pnas.1212246109

SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing
journal, October 2015

  • Guo, Xiaoge; Lehner, Kevin; O’Connell, Karen
  • G3: Genes|Genomes|Genetics, Vol. 5, Issue 12
  • DOI: 10.1534/g3.115.023317

Origins of human mitochondrial point mutations as DNA polymerase γ-mediated errors
journal, July 2006

  • Zheng, Weiming; Khrapko, Konstantin; Coller, Hilary A.
  • Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 599, Issue 1-2
  • DOI: 10.1016/j.mrfmmm.2005.12.012

Fidelity of DNA polymerases in DNA amplification.
journal, December 1989

  • Keohavong, P.; Thilly, W. G.
  • Proceedings of the National Academy of Sciences, Vol. 86, Issue 23
  • DOI: 10.1073/pnas.86.23.9253

Detection and quantification of rare mutations with massively parallel sequencing
journal, May 2011

  • Kinde, I.; Wu, J.; Papadopoulos, N.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 23
  • DOI: 10.1073/pnas.1105422108

High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing
journal, November 2013

  • Lou, D. I.; Hussmann, J. A.; McBee, R. M.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 49
  • DOI: 10.1073/pnas.1319590110

Fidelity of DNA polymerases in DNA amplification.
journal, December 1989

  • Keohavong, P.; Thilly, W. G.
  • Proceedings of the National Academy of Sciences, Vol. 86, Issue 23
  • DOI: 10.1073/pnas.86.23.9253

A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates
journal, January 2006

  • Jarosz, Daniel F.; Godoy, Veronica G.; Delaney, James C.
  • Nature, Vol. 439, Issue 7073
  • DOI: 10.1038/nature04318

DNA polymerase ε and its roles in genome stability: ROLES OF DNA Pol ε IN GENOME STABILITY
journal, May 2014

  • Henninger, Erin E.; Pursell, Zachary F.
  • IUBMB Life, Vol. 66, Issue 5
  • DOI: 10.1002/iub.1276

Analyzing fidelity of DNA polymerases
book, January 1995

Mutational spectrometry without phenotypic selection: human mitochondrial DNA
journal, February 1997

  • Khrapko, K.; Coller, H.; Andre, P.
  • Nucleic Acids Research, Vol. 25, Issue 4
  • DOI: 10.1093/nar/25.4.685

A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates
journal, January 2006

  • Jarosz, Daniel F.; Godoy, Veronica G.; Delaney, James C.
  • Nature, Vol. 439, Issue 7073
  • DOI: 10.1038/nature04318

Evolving Views of DNA Replication (In)Fidelity
journal, January 2009

Direct cloning and sequence analysis of enzymatically amplified genomic sequences
journal, September 1986

Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing
journal, September 2012

  • Lee, H.; Popodi, E.; Tang, H.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 41
  • DOI: 10.1073/pnas.1210309109

Mutational spectrometry without phenotypic selection: human mitochondrial DNA
journal, February 1997

  • Khrapko, K.; Coller, H.; Andre, P.
  • Nucleic Acids Research, Vol. 25, Issue 4
  • DOI: 10.1093/nar/25.4.685

Origins of human mitochondrial point mutations as DNA polymerase γ-mediated errors
journal, July 2006

  • Zheng, Weiming; Khrapko, Konstantin; Coller, Hilary A.
  • Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 599, Issue 1-2
  • DOI: 10.1016/j.mrfmmm.2005.12.012

Detection of ultra-rare mutations by next-generation sequencing
journal, August 2012

  • Schmitt, M. W.; Kennedy, S. R.; Salk, J. J.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 36
  • DOI: 10.1073/pnas.1208715109

SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing
journal, October 2015

  • Guo, Xiaoge; Lehner, Kevin; O’Connell, Karen
  • G3: Genes|Genomes|Genetics, Vol. 5, Issue 12
  • DOI: 10.1534/g3.115.023317

Characterization of Escherichia coli Translesion Synthesis Polymerases and Their Accessory Factors
book, January 2006

High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing
journal, November 2013

  • Lou, D. I.; Hussmann, J. A.; McBee, R. M.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 49
  • DOI: 10.1073/pnas.1319590110

Assays for Determining Lesion Bypass Efficiency and Mutagenicity of Site-Specific DNA Lesions In Vivo
book, January 2006

DNA polymerases and cancer
journal, January 2011

  • Lange, Sabine S.; Takata, Kei-ichi; Wood, Richard D.
  • Nature Reviews Cancer, Vol. 11, Issue 2
  • DOI: 10.1038/nrc2998

MUSCLE: multiple sequence alignment with high accuracy and high throughput
journal, March 2004

  • Edgar, R. C.
  • Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797
  • DOI: 10.1093/nar/gkh340

Recent Developments in Computer-Assisted Analysis of Mixtures
journal, June 1998

  • Bohning, Dankmar; Dietz, Ekkehart; Schlattmann, Peter
  • Biometrics, Vol. 54, Issue 2
  • DOI: 10.2307/3109760

Direct cloning and sequence analysis of enzymatically amplified genomic sequences
journal, September 1986

Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing
journal, September 2012

  • Lee, H.; Popodi, E.; Tang, H.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 41
  • DOI: 10.1073/pnas.1210309109

Evolving Views of DNA Replication (In)Fidelity
journal, January 2009

Error-Prone Polymerase, DNA Polymerase IV, Is Responsible for Transient Hypermutation during Adaptive Mutation in Escherichia coli
journal, June 2003

Streisinger Revisited: DNA Synthesis Errors Mediated by Substrate Misalignments
journal, January 2000

  • Bebenek, K.; Kunkel, T. A.
  • Cold Spring Harbor Symposia on Quantitative Biology, Vol. 65, Issue 0
  • DOI: 10.1101/sqb.2000.65.81

Targeted single molecule mutation detection with massively parallel sequencing
journal, September 2015

  • Gregory, Mark T.; Bertout, Jessica A.; Ericson, Nolan G.
  • Nucleic Acids Research, Vol. 44, Issue 3
  • DOI: 10.1093/nar/gkv915

Retroviral mutation rates and reverse transcriptase fidelity
journal, January 2003

  • Pathak, Vinay K.
  • Frontiers in Bioscience, Vol. 8, Issue 4
  • DOI: 10.2741/957

The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I.
journal, August 1990

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    Revealing the Complexities of Metabarcoding with a Diverse Arthropod Mock Community
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    In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR
    journal, August 2017

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    High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing
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