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

Ellegaard KM, Klasson L, Andersson SG - PLoS ONE (2013)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

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

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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Related in: MedlinePlus

Heat map of Kendalls tau correlation coefficients.Pairwise correlations between the coverage patterns (averaged in 1 kb windows) in Illumina data sets of MDA samples taken from dilution series of intact bacterial cells (cells2-cells5) and genomic DNA (gDNA1-gDNA8), as detailed in Tables 1 and 2, respectively. Sample "control 1" refers to the unamplified Illumina data set obtained from the re-sequencing of the B. australis in this study. Sample "control 2" refers to the unamplified Illumina data set obtained from the previously published B. australis genome.
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pone-0082319-g003: Heat map of Kendalls tau correlation coefficients.Pairwise correlations between the coverage patterns (averaged in 1 kb windows) in Illumina data sets of MDA samples taken from dilution series of intact bacterial cells (cells2-cells5) and genomic DNA (gDNA1-gDNA8), as detailed in Tables 1 and 2, respectively. Sample "control 1" refers to the unamplified Illumina data set obtained from the re-sequencing of the B. australis in this study. Sample "control 2" refers to the unamplified Illumina data set obtained from the previously published B. australis genome.

Mentions: To evaluate the similarity in patterns between samples, we calculated the correlation of coverage in 1000 bp windows between all possible pairwise combinations of samples using Kendalls Tau (Figure 3), which is a measure of the correlation between samples that do not follow a normal distribution [32]. Basically, all datapoints in each sample were ranked and the agreement between the ranking orders of two samples at a time was calculated. For example, if region A has a higher coverage than region B in both samples, the ranks are in agreement. We found the strongest correlations in amplification bias pattern between samples with similar quantities of template DNA. For example, sample “gDNA5” had a slightly higher correlation to sample “gDNA6” than to “gDNA1”. The highest correlation value was between the samples "cells2" and “cells3”, which were prepared from the largest number of cells of the samples analyzed here (see Figure 1F for a comparison of the patterns).


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

Ellegaard KM, Klasson L, Andersson SG - PLoS ONE (2013)

Heat map of Kendalls tau correlation coefficients.Pairwise correlations between the coverage patterns (averaged in 1 kb windows) in Illumina data sets of MDA samples taken from dilution series of intact bacterial cells (cells2-cells5) and genomic DNA (gDNA1-gDNA8), as detailed in Tables 1 and 2, respectively. Sample "control 1" refers to the unamplified Illumina data set obtained from the re-sequencing of the B. australis in this study. Sample "control 2" refers to the unamplified Illumina data set obtained from the previously published B. australis genome.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842359&req=5

pone-0082319-g003: Heat map of Kendalls tau correlation coefficients.Pairwise correlations between the coverage patterns (averaged in 1 kb windows) in Illumina data sets of MDA samples taken from dilution series of intact bacterial cells (cells2-cells5) and genomic DNA (gDNA1-gDNA8), as detailed in Tables 1 and 2, respectively. Sample "control 1" refers to the unamplified Illumina data set obtained from the re-sequencing of the B. australis in this study. Sample "control 2" refers to the unamplified Illumina data set obtained from the previously published B. australis genome.
Mentions: To evaluate the similarity in patterns between samples, we calculated the correlation of coverage in 1000 bp windows between all possible pairwise combinations of samples using Kendalls Tau (Figure 3), which is a measure of the correlation between samples that do not follow a normal distribution [32]. Basically, all datapoints in each sample were ranked and the agreement between the ranking orders of two samples at a time was calculated. For example, if region A has a higher coverage than region B in both samples, the ranks are in agreement. We found the strongest correlations in amplification bias pattern between samples with similar quantities of template DNA. For example, sample “gDNA5” had a slightly higher correlation to sample “gDNA6” than to “gDNA1”. The highest correlation value was between the samples "cells2" and “cells3”, which were prepared from the largest number of cells of the samples analyzed here (see Figure 1F for a comparison of the patterns).

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus