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

PLoS One

Department of Molecular Evolution, Cell and Molecular Biology, Science for Life Laboratory, Biomedical Centre, Uppsala University, Uppsala, Sweden.

Published: August 2014

AI Article Synopsis

  • The multiple displacement amplification (MDA) method has significantly advanced the study of uncultured bacteria but presents issues like uneven DNA amplification and contamination.
  • Testing MDA with various dilutions of DNA from Bartonella australis revealed that as the template DNA amount decreases, problems like amplification bias and chimeric reads worsen.
  • Using multiple cells instead of single cells for amplification can reduce these artifacts, providing valuable insights for researchers investigating the genomes of endosymbionts and other hard-to-cultivate bacteria.

Article 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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842359PMC
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082319PLOS

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