Department of Molecular Evolution, Cell and Molecular Biology, Science for Life Laboratory, Biomedical Centre, Uppsala University, Uppsala, Sweden.
PLoS One. 2013 Nov 27;8(11):e82319. doi: 10.1371/journal.pone.0082319. eCollection 2013.
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.
多重置换扩增方法彻底改变了对未培养细菌的基因组研究，因为从这些细菌中提取足够数量的纯 DNA 用于下一代测序是具有挑战性的。然而，该方法存在问题，因为它会不均匀地扩增靶 DNA，诱导嵌合读取的形成，并扩增污染 DNA。在这里，我们使用从培养的内共生菌 Bartonella australis 中提取的基因组 DNA 和完整细胞的连续稀释液测试了多重置换扩增方法的重现性。扩增的 DNA 用 Illumina 测序技术进行测序，并将结果与本研究中未扩增 DNA 的序列数据以及之前发表的基因组项目进行比较。我们表明，随着模板 DNA 量的减少，诸如扩增偏倚的程度、嵌合读取的百分比和污染 DNA 的相对分数等伪影急剧增加。对于具有较高模板 DNA 量的样本，可以重复获得读取覆盖模式，这表明偏差是非随机的和基因组特异性的。对以前从克隆内共生体群体扩增后获得的序列数据的重新分析证实了这些预测。我们得出结论，通过使用多个细胞作为扩增的模板，可以缓解或大大减少与使用多重置换扩增方法相关的许多伪影。这些发现对于研究内共生体和其他未培养细菌基因组的研究人员特别有用，因为可以从这些细菌中分离出一小部分克隆细胞。
