A high-throughput assay for quantitative measurement of PCR errors

Abstract 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...

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Autores principales:	Dmitriy A. Shagin, Irina A. Shagina, Andrew R. Zaretsky, Ekaterina V. Barsova, Ilya V. Kelmanson, Sergey Lukyanov, Dmitriy M. Chudakov, Mikhail Shugay
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c1694952e05043f282fa23db7a93a02d
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spelling	oai:doaj.org-article:c1694952e05043f282fa23db7a93a02d2021-12-02T15:06:16ZA high-throughput assay for quantitative measurement of PCR errors10.1038/s41598-017-02727-82045-2322https://doaj.org/article/c1694952e05043f282fa23db7a93a02d2017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-02727-8https://doaj.org/toc/2045-2322Abstract 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.Dmitriy A. ShaginIrina A. ShaginaAndrew R. ZaretskyEkaterina V. BarsovaIlya V. KelmansonSergey LukyanovDmitriy M. ChudakovMikhail ShugayNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Dmitriy A. Shagin Irina A. Shagina Andrew R. Zaretsky Ekaterina V. Barsova Ilya V. Kelmanson Sergey Lukyanov Dmitriy M. Chudakov Mikhail Shugay A high-throughput assay for quantitative measurement of PCR errors
description	Abstract 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.
format	article
author	Dmitriy A. Shagin Irina A. Shagina Andrew R. Zaretsky Ekaterina V. Barsova Ilya V. Kelmanson Sergey Lukyanov Dmitriy M. Chudakov Mikhail Shugay
author_facet	Dmitriy A. Shagin Irina A. Shagina Andrew R. Zaretsky Ekaterina V. Barsova Ilya V. Kelmanson Sergey Lukyanov Dmitriy M. Chudakov Mikhail Shugay
author_sort	Dmitriy A. Shagin
title	A high-throughput assay for quantitative measurement of PCR errors
title_short	A high-throughput assay for quantitative measurement of PCR errors
title_full	A high-throughput assay for quantitative measurement of PCR errors
title_fullStr	A high-throughput assay for quantitative measurement of PCR errors
title_full_unstemmed	A high-throughput assay for quantitative measurement of PCR errors
title_sort	high-throughput assay for quantitative measurement of pcr errors
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/c1694952e05043f282fa23db7a93a02d
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A high-throughput assay for quantitative measurement of PCR errors

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