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Nucleic Acids Res. 2016 Apr 20;44(7):e66. doi: 10.1093/nar/gkv1493. Epub 2015 Dec 23.

Enhanced sequencing coverage with digital droplet multiple displacement amplification.

Author information

1
Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, USA Berkeley/UCSF Graduate Program in Bioengineering, University of California, San Francisco, CA 94158, USA.
2
Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, USA Berkeley/UCSF Graduate Program in Bioengineering, University of California, San Francisco, CA 94158, USA adam.abate@ucsf.edu.

Abstract

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.

PMID:
26704978
PMCID:
PMC4838355
DOI:
10.1093/nar/gkv1493
[Indexed for MEDLINE]
Free PMC Article

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