Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells

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Abstract
Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-μl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells. It is often challenging to manipulate or analyze the genetic material or genome of an individual cell. Biochemical DNA amplification technologies can be used to make many copies of the genome from a single cell, and in this paper we investigated how well such amplification works as a function of the reaction volume. We found that single-cell genome amplification in nanoliter volumes is much more effective than in microliter volumes, providing better representation of the starting genome with less bias in the product. It should therefore be possible to obtain high-quality genome sequences from single cells. This is useful because very few microbes can be obtained in pure culture, and are therefore only amenable to single-cell analysis.