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The efficacy and further functional advantages of random-base molecular barcodes for absolute and digital quantification of nucleic acid molecules

Accurate quantification of biomolecules in system-wide measurements is in high demand, especially for systems with limited sample amounts such as single cells. Because of this, digital quantification of nucleic acid molecules using molecular barcodes has been developed, making, e.g., transcriptome a...

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Autores principales: Ogawa, Taisaku, Kryukov, Kirill, Imanishi, Tadashi, Shiroguchi, Katsuyuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648891/
https://www.ncbi.nlm.nih.gov/pubmed/29051542
http://dx.doi.org/10.1038/s41598-017-13529-3
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author Ogawa, Taisaku
Kryukov, Kirill
Imanishi, Tadashi
Shiroguchi, Katsuyuki
author_facet Ogawa, Taisaku
Kryukov, Kirill
Imanishi, Tadashi
Shiroguchi, Katsuyuki
author_sort Ogawa, Taisaku
collection PubMed
description Accurate quantification of biomolecules in system-wide measurements is in high demand, especially for systems with limited sample amounts such as single cells. Because of this, digital quantification of nucleic acid molecules using molecular barcodes has been developed, making, e.g., transcriptome analysis highly reproducible and quantitative. This counting scheme was shown to work using sequence-restricted barcodes, and non-sequence-restricted (random-base) barcodes that may provide a much higher dynamic range at significantly lower cost have been widely used. However, the efficacy of random-base barcodes is significantly affected by base changes due to amplification and/or sequencing errors and has not been investigated experimentally or quantitatively. Here, we show experimentally that random-base barcodes enable absolute and digital quantification of DNA molecules with high dynamic range (from one to more than 10(4), potentially up to 10(15) molecules) conditional on our barcode design and variety, a certain range of sequencing depths, and computational analyses. Moreover, we quantitatively show further functional advantages of the molecular barcodes: the molecular barcodes enable one to find contaminants and misidentifications of target sequences. Our scheme here may be generally used to confirm that the digital quantification works in each platform.
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spelling pubmed-56488912017-10-26 The efficacy and further functional advantages of random-base molecular barcodes for absolute and digital quantification of nucleic acid molecules Ogawa, Taisaku Kryukov, Kirill Imanishi, Tadashi Shiroguchi, Katsuyuki Sci Rep Article Accurate quantification of biomolecules in system-wide measurements is in high demand, especially for systems with limited sample amounts such as single cells. Because of this, digital quantification of nucleic acid molecules using molecular barcodes has been developed, making, e.g., transcriptome analysis highly reproducible and quantitative. This counting scheme was shown to work using sequence-restricted barcodes, and non-sequence-restricted (random-base) barcodes that may provide a much higher dynamic range at significantly lower cost have been widely used. However, the efficacy of random-base barcodes is significantly affected by base changes due to amplification and/or sequencing errors and has not been investigated experimentally or quantitatively. Here, we show experimentally that random-base barcodes enable absolute and digital quantification of DNA molecules with high dynamic range (from one to more than 10(4), potentially up to 10(15) molecules) conditional on our barcode design and variety, a certain range of sequencing depths, and computational analyses. Moreover, we quantitatively show further functional advantages of the molecular barcodes: the molecular barcodes enable one to find contaminants and misidentifications of target sequences. Our scheme here may be generally used to confirm that the digital quantification works in each platform. Nature Publishing Group UK 2017-10-19 /pmc/articles/PMC5648891/ /pubmed/29051542 http://dx.doi.org/10.1038/s41598-017-13529-3 Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Ogawa, Taisaku
Kryukov, Kirill
Imanishi, Tadashi
Shiroguchi, Katsuyuki
The efficacy and further functional advantages of random-base molecular barcodes for absolute and digital quantification of nucleic acid molecules
title The efficacy and further functional advantages of random-base molecular barcodes for absolute and digital quantification of nucleic acid molecules
title_full The efficacy and further functional advantages of random-base molecular barcodes for absolute and digital quantification of nucleic acid molecules
title_fullStr The efficacy and further functional advantages of random-base molecular barcodes for absolute and digital quantification of nucleic acid molecules
title_full_unstemmed The efficacy and further functional advantages of random-base molecular barcodes for absolute and digital quantification of nucleic acid molecules
title_short The efficacy and further functional advantages of random-base molecular barcodes for absolute and digital quantification of nucleic acid molecules
title_sort efficacy and further functional advantages of random-base molecular barcodes for absolute and digital quantification of nucleic acid molecules
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648891/
https://www.ncbi.nlm.nih.gov/pubmed/29051542
http://dx.doi.org/10.1038/s41598-017-13529-3
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