Enhanced sequencing coverage with digital droplet multiple displacement amplification

Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significan...

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Autores principales: Sidore, Angus M., Lan, Freeman, Lim, Shaun W., Abate, Adam R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838355/
https://www.ncbi.nlm.nih.gov/pubmed/26704978
http://dx.doi.org/10.1093/nar/gkv1493
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author Sidore, Angus M.
Lan, Freeman
Lim, Shaun W.
Abate, Adam R.
author_facet Sidore, Angus M.
Lan, Freeman
Lim, Shaun W.
Abate, Adam R.
author_sort Sidore, Angus M.
collection PubMed
description Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing.
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spelling pubmed-48383552016-04-21 Enhanced sequencing coverage with digital droplet multiple displacement amplification Sidore, Angus M. Lan, Freeman Lim, Shaun W. Abate, Adam R. Nucleic Acids Res Methods Online Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing. Oxford University Press 2016-04-20 2015-12-23 /pmc/articles/PMC4838355/ /pubmed/26704978 http://dx.doi.org/10.1093/nar/gkv1493 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Methods Online
Sidore, Angus M.
Lan, Freeman
Lim, Shaun W.
Abate, Adam R.
Enhanced sequencing coverage with digital droplet multiple displacement amplification
title Enhanced sequencing coverage with digital droplet multiple displacement amplification
title_full Enhanced sequencing coverage with digital droplet multiple displacement amplification
title_fullStr Enhanced sequencing coverage with digital droplet multiple displacement amplification
title_full_unstemmed Enhanced sequencing coverage with digital droplet multiple displacement amplification
title_short Enhanced sequencing coverage with digital droplet multiple displacement amplification
title_sort enhanced sequencing coverage with digital droplet multiple displacement amplification
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838355/
https://www.ncbi.nlm.nih.gov/pubmed/26704978
http://dx.doi.org/10.1093/nar/gkv1493
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