Testing the reproducibility of multiple displacement amplification on genomes of clonal endosymbiont populations

PLoS One. 2013 Nov 27;8(11):e82319. doi: 10.1371/journal.pone.0082319. eCollection 2013.

Abstract

The multiple displacement amplification method has revolutionized genomic studies of uncultured bacteria, where the extraction of pure DNA in sufficient quantity for next-generation sequencing is challenging. However, the method is problematic in that it amplifies the target DNA unevenly, induces the formation of chimeric reads and also amplifies contaminating DNA. Here, we have tested the reproducibility of the multiple displacement amplification method using serial dilutions of extracted genomic DNA and intact cells from the cultured endosymbiont Bartonella australis. The amplified DNA was sequenced with the Illumina sequencing technology, and the results were compared to sequence data obtained from unamplified DNA in this study as well as from a previously published genome project. We show that artifacts such as the extent of the amplification bias, the percentage of chimeric reads and the relative fraction of contaminating DNA increase dramatically for the smallest amounts of template DNA. The pattern of read coverage was reproducibly obtained for samples with higher amounts of template DNA, suggesting that the bias is non-random and genome-specific. A re-analysis of previously published sequence data obtained after amplification from clonal endosymbiont populations confirmed these predictions. We conclude that many of the artifacts associated with the use of the multiple displacement amplification method can be alleviated or much reduced by using multiple cells as the template for the amplification. These findings should be particularly useful for researchers studying the genomes of endosymbionts and other uncultured bacteria, for which a small clonal population of cells can be isolated.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bartonella / genetics
  • DNA, Bacterial / genetics
  • Genome, Bacterial*
  • Reproducibility of Results
  • Symbiosis*

Substances

  • DNA, Bacterial

Grants and funding

The authors have been supported by grants to LK from the Swedish Research Council for Agricultural Sciences, to SGEA from the European Union, the Swedish Research Council and the Knut and Alice Wallenberg Foundation and to LK and SGEA from the Science for Life Laboratory. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.