A high-throughput assay for quantitative measurement of PCR
errors

J 2017

A high-throughput assay for quantitative measurement of PCR errors

SHAGIN, D.A., I.A. SHAGINA, A.R. ZARETSKY, E.V. BARSOVA, I.V. KELMANSON et. al.

Basic information

Original name

A high-throughput assay for quantitative measurement of PCR errors

Authors

SHAGIN, D.A. (643 Russian Federation), I.A. SHAGINA (643 Russian Federation), A.R. ZARETSKY (643 Russian Federation), E.V. BARSOVA (643 Russian Federation), I.V. KELMANSON (643 Russian Federation), S. LUKYANOV (643 Russian Federation), Dmitriy CHUDAKOV (643 Russian Federation, guarantor, belonging to the institution) and Mikhail SHUGAY (643 Russian Federation, belonging to the institution)

Edition

Scientific reports, LONDON, NATURE PUBLISHING GROUP, 2017, 2045-2322

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10609 Biochemical research methods

Country of publisher

United Kingdom of Great Britain and Northern Ireland

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 4.122

RIV identification code

RIV/00216224:14740/17:00100340

Organization unit

Central European Institute of Technology

DOI

http://dx.doi.org/10.1038/s41598-017-02727-8

UT WoS

000402515800036

Keywords in English

DRUG-RESISTANCE MUTATIONS; DNA-POLYMERASE; RARE MUTATIONS; FIDELITY; AMPLIFICATION; SPECIFICITY

Abstract

V originále

The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.

Links

LQ1601, research and development project

Name: CEITEC 2020 (Acronym: CEITEC2020)

Investor: Ministry of Education, Youth and Sports of the CR

Citovat

SHAGIN, D.A., I.A. SHAGINA, A.R. ZARETSKY, E.V. BARSOVA, I.V. KELMANSON, S. LUKYANOV, Dmitriy CHUDAKOV and Mikhail SHUGAY. A high-throughput assay for quantitative measurement of PCR errors. Scientific reports. LONDON: NATURE PUBLISHING GROUP, 2017, vol. 7, JUN, p. 2718-2728. ISSN 2045-2322. Available from: https://dx.doi.org/10.1038/s41598-017-02727-8.

@article{1411035,
   author = {Shagin, D.A. and Shagina, I.A. and Zaretsky, A.R. and Barsova, E.V. and Kelmanson, I.V. and Lukyanov, S. and Chudakov, Dmitriy and Shugay, Mikhail},
   article_location = {LONDON},
   article_number = {JUN},
   doi = {http://dx.doi.org/10.1038/s41598-017-02727-8},
   keywords = {DRUG-RESISTANCE MUTATIONS; DNA-POLYMERASE; RARE MUTATIONS; FIDELITY; AMPLIFICATION; SPECIFICITY},
   language = {eng},
   issn = {2045-2322},
   journal = {Scientific reports},
   title = {A high-throughput assay for quantitative measurement of PCR errors},
   url = {https://www.nature.com/articles/s41598-017-02727-8.pdf},
   volume = {7},
   year = {2017}
}

TY  - JOUR
ID  - 1411035
AU  - Shagin, D.A. - Shagina, I.A. - Zaretsky, A.R. - Barsova, E.V. - Kelmanson, I.V. - Lukyanov, S. - Chudakov, Dmitriy - Shugay, Mikhail
PY  - 2017
TI  - A high-throughput assay for quantitative measurement of PCR errors
JF  - Scientific reports
VL  - 7
IS  - JUN
SP  - 2718
EP  - 2718
PB  - NATURE PUBLISHING GROUP
SN  - 20452322
KW  - DRUG-RESISTANCE MUTATIONS
KW  - DNA-POLYMERASE
KW  - RARE MUTATIONS
KW  - FIDELITY
KW  - AMPLIFICATION
KW  - SPECIFICITY
UR  - https://www.nature.com/articles/s41598-017-02727-8.pdf
L2  - https://www.nature.com/articles/s41598-017-02727-8.pdf
N2  - The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.
ER  -

SHAGIN, D.A., I.A. SHAGINA, A.R. ZARETSKY, E.V. BARSOVA, I.V. KELMANSON, S. LUKYANOV, Dmitriy CHUDAKOV and Mikhail SHUGAY. A high-throughput assay for quantitative measurement of PCR errors. \textit{Scientific reports}. LONDON: NATURE PUBLISHING GROUP, 2017, vol.~7, JUN, p.~2718-2728. ISSN~2045-2322. Available from: https://dx.doi.org/10.1038/s41598-017-02727-8.

Detailed Information on Publication Record