Genotyping of genetically monomorphic bacteria: DNA sequencing in Mycobacterium tuberculosis highlights the limitations of current methodologies.
Genotyping of genetically monomorphic bacteria: DNA sequencing in Mycobacterium tuberculosis highlights the limitations of current methodologies.
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DOI：
10.1371/journal.pone.0007815
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发表时间：
2009-11-12
期刊：
影响因子：
3.7
通讯作者：
Gagneux S
中科院分区：
文献类型：
--
作者：
Comas I;Homolka S;Niemann S;Gagneux S
由于基因单态细菌病原体几乎没有 DNA 序列多样性，目前用于研究这些生物体流行病学的大多数基因分型技术都是基于移动或重复的遗传元件。这些细菌中常用的分子标记包括成簇调节短回文重复序列 (CRISPR) 和可变数量串联重复序列 (VNTR)。这些方法也越来越多地应用于系统发育和群体遗传学研究。使用结核分枝杆菌复合体 (MTBC) 作为模型，我们评估了基于 CRISPR 和 VNTR 的基因分型的系统发育准确性，在 MTBC 中分别称为 spoligotyping 和分枝杆菌散布重复单位 (MIRU)-VNTR 分型。我们使用来自全球菌株库的 89 个编码基因的完整 DNA 序列作为黄金标准。我们的结果表明，无论使用何种系统发育方法，从这些多位点序列数据衍生的系统发育树都高度一致且统计稳健。相比之下，从 spoligotyping 或 15-位点-MIRU-VNTR 推断出的相应系统发育与基于序列的树不一致。尽管 24-loci-MIRU-VNTR 表现更好，但仍然无法检测所有菌株谱系。 DNA 序列数据显示几乎没有同质性，但 spoligotyping 和 MIRU-VNTR 的情况恰恰相反，这与趋同进化的高速率和由这些标记定义的系统发育分组获得的低统计支持一致。我们的结果还表明，标准 24 个 MIRU-VNTR 位点的区分能力因菌株谱系而异。综上所述，我们的研究结果表明，MTBC 中的菌株谱系应基于系统发育上稳健的标记（例如单核苷酸多态性或大序列多态性）进行定义，并且出于流行病学目的，MIRU-VNTR 基因座应以谱系依赖性方式使用。我们的发现对其他遗传单态细菌的菌株分型具有影响。
Because genetically monomorphic bacterial pathogens harbour little DNA sequence diversity, most current genotyping techniques used to study the epidemiology of these organisms are based on mobile or repetitive genetic elements. Molecular markers commonly used in these bacteria include Clustered Regulatory Short Palindromic Repeats (CRISPR) and Variable Number Tandem Repeats (VNTR). These methods are also increasingly being applied to phylogenetic and population genetic studies. Using the Mycobacterium tuberculosis complex (MTBC) as a model, we evaluated the phylogenetic accuracy of CRISPR- and VNTR-based genotyping, which in MTBC are known as spoligotyping and Mycobacterial Interspersed Repetitive Units (MIRU)-VNTR-typing, respectively. We used as a gold standard the complete DNA sequences of 89 coding genes from a global strain collection. Our results showed that phylogenetic trees derived from these multilocus sequence data were highly congruent and statistically robust, irrespective of the phylogenetic methods used. By contrast, corresponding phylogenies inferred from spoligotyping or 15-loci-MIRU-VNTR were incongruent with respect to the sequence-based trees. Although 24-loci-MIRU-VNTR performed better, it was still unable to detect all strain lineages. The DNA sequence data showed virtually no homoplasy, but the opposite was true for spoligotyping and MIRU-VNTR, which was consistent with high rates of convergent evolution and the low statistical support obtained for phylogenetic groupings defined by these markers. Our results also revealed that the discriminatory power of the standard 24 MIRU-VNTR loci varied by strain lineage. Taken together, our findings suggest strain lineages in MTBC should be defined based on phylogenetically robust markers such as single nucleotide polymorphisms or large sequence polymorphisms, and that for epidemiological purposes, MIRU-VNTR loci should be used in a lineage-dependent manner. Our findings have implications for strain typing in other genetically monomorphic bacteria.
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影响因子：
3.2
作者：
Filliol, I;Motiwala, AS;Alland, D
通讯作者：
Alland, D
影响因子：
6.4
作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
AKAIKE, H
DOI：
10.1073/pnas.0408026101
发表时间：
2004-12-21
影响因子：
11.1
作者：
Achtman, M;Morelli, G;Keim, P
通讯作者：
Keim, P