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Testing the Reproducibility of Multiple Displacement Amplification on Genomes of Clonal Endosymbiont Populations
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, e82319- p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2013. Vol. 8, no 11, e82319- p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-214040DOI: 10.1371/journal.pone.0082319ISI: 000327652100111OAI: oai:DiVA.org:uu-214040DiVA: diva2:684072
Available from: 2014-01-07 Created: 2014-01-07 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Genome Evolution and Niche Differentiation of Bacterial Endosymbionts
Open this publication in new window or tab >>Genome Evolution and Niche Differentiation of Bacterial Endosymbionts
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Most animals contain chronic microbial infections that inflict no harm on their hosts. Recently, the gut microflora of humans and other animals have been characterized. However, little is known about the forces that shape the diversity of these bacterial communities. In this work, comparative genomics was used to investigate the evolutionary dynamics of host-adapted bacterial communities, using Wolbachia infecting arthropods and Lactobacteria infecting bees as the main model systems.

Wolbachia are maternally inherited bacteria that cause reproductive disorders in arthropods, such as feminization, male killing and parthenogenesis. These bacteria are difficult to study because they cannot be cultivated outside their hosts. We have developed a novel protocol employing multiple displacement amplification to isolate and sequence their genomes. Taxonomically, Wolbachia is classified into different supergroups. We have sequenced the genomes of Wolbachia strain wHa and wNo that belong to supergroup A and B, respectively, and are present as a double-infection in the fruit-fly Drosophila simulans. Together with previously published genomes, a supergroup comparison of strains belonging to supergroups A and B indicated rampant homologous recombination between strains that belong to the same supergroup but were isolated from different hosts. In contrast, we observed little recombination between strains of different supergroups that infect the same host.

Likewise, phylogenetically distinct members of Lactic acid bacteria co-exist in the gut of the honeybee, Apis mellifera, without transfer of genes between phylotypes. Nor did we find any evidence of co-diversification between symbionts and hosts, as inferred from a study of 13 genomes of Lactobacillus kunkeei isolated from diverse bee species and different geographic origins. Although Lactobacillus kunkeii is the most frequently isolated strain from the honey stomach, we hypothesize that the primary niche is the beebread where the bacteria are likely to contribute to the fermentation process.

In the human gut, the microbial community has been shown to interact with the immune system, and likewise the microbial communities associated with insects are thought to affect the health of their host. Therefore, a better understanding of the role and evolution of endosymbiotic communities is important for developing strategies to control the health of their hosts.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1121
Keyword
niche, habitat, endosymbiont, gut microbiome, honey bee, Wolbachia, comparative, genomics
National Category
Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-217724 (URN)978-91-554-8872-7 (ISBN)
External cooperation:
Public defence
2014-03-21, B42, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2014-02-27 Created: 2014-02-04 Last updated: 2016-08-26Bibliographically approved

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Ellegaard, Kirsten MarenKlasson, LisaAndersson, Siv G. E.

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