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Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding
The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq, and in the context of analysis of T and B cell receptor repertoires. In this stud...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602216/ https://www.ncbi.nlm.nih.gov/pubmed/26459131 http://dx.doi.org/10.1038/srep14629 |
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author | Best, Katharine Oakes, Theres Heather, James M. Shawe-Taylor, John Chain, Benny |
author_facet | Best, Katharine Oakes, Theres Heather, James M. Shawe-Taylor, John Chain, Benny |
author_sort | Best, Katharine |
collection | PubMed |
description | The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq, and in the context of analysis of T and B cell receptor repertoires. In this study, we combine DNA barcoding with HTS to quantify PCR output from individual target molecules. We develop computational tools that simulate both the PCR branching process itself, and the subsequent subsampling which typically occurs during HTS sequencing. We explore the influence of different types of heterogeneity on sequencing output, and compare them to experimental results where the efficiency of amplification is measured by barcodes uniquely identifying each molecule of starting template. Our results demonstrate that the PCR process introduces substantial amplification heterogeneity, independent of primer sequence and bulk experimental conditions. This heterogeneity can be attributed both to inherited differences between different template DNA molecules, and the inherent stochasticity of the PCR process. The results demonstrate that PCR heterogeneity arises even when reaction and substrate conditions are kept as constant as possible, and therefore single molecule barcoding is essential in order to derive reproducible quantitative results from any protocol combining PCR with HTS. |
format | Online Article Text |
id | pubmed-4602216 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-46022162015-10-23 Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding Best, Katharine Oakes, Theres Heather, James M. Shawe-Taylor, John Chain, Benny Sci Rep Article The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq, and in the context of analysis of T and B cell receptor repertoires. In this study, we combine DNA barcoding with HTS to quantify PCR output from individual target molecules. We develop computational tools that simulate both the PCR branching process itself, and the subsequent subsampling which typically occurs during HTS sequencing. We explore the influence of different types of heterogeneity on sequencing output, and compare them to experimental results where the efficiency of amplification is measured by barcodes uniquely identifying each molecule of starting template. Our results demonstrate that the PCR process introduces substantial amplification heterogeneity, independent of primer sequence and bulk experimental conditions. This heterogeneity can be attributed both to inherited differences between different template DNA molecules, and the inherent stochasticity of the PCR process. The results demonstrate that PCR heterogeneity arises even when reaction and substrate conditions are kept as constant as possible, and therefore single molecule barcoding is essential in order to derive reproducible quantitative results from any protocol combining PCR with HTS. Nature Publishing Group 2015-10-13 /pmc/articles/PMC4602216/ /pubmed/26459131 http://dx.doi.org/10.1038/srep14629 Text en Copyright © 2015, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Article Best, Katharine Oakes, Theres Heather, James M. Shawe-Taylor, John Chain, Benny Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding |
title | Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding |
title_full | Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding |
title_fullStr | Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding |
title_full_unstemmed | Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding |
title_short | Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding |
title_sort | computational analysis of stochastic heterogeneity in pcr amplification efficiency revealed by single molecule barcoding |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602216/ https://www.ncbi.nlm.nih.gov/pubmed/26459131 http://dx.doi.org/10.1038/srep14629 |
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