Using high throughput sequencing to explore the biodiversity in oral bacterial communities.
Using high throughput sequencing to explore the biodiversity in oral bacterial communities.
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DOI：
10.1111/j.2041-1014.2012.00642.x
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发表时间：
2012-06
影响因子：
3.7
通讯作者：
Strausbaugh LD
中科院分区：
文献类型：
--
作者：
Diaz PI;Dupuy AK;Abusleme L;Reese B;Obergfell C;Choquette L;Dongari-Bagtzoglou A;Peterson DE;Terzi E;Strausbaugh LD
16S核糖体RNA基因扩增子的高通量测序是表征口腔细菌群落的一种具有成本效益的方法。然而，在进行大规模研究之前，有必要了解技术相关的限制和口腔生态系统的内在变异性。在这项工作中，我们评估了偏见的物种代表性使用体外组装模拟社区的口腔细菌。然后，我们对五名健康受试者唾液和颊粘膜中的细菌群落进行了表征，以研究高通量测序在揭示其多样性和生物地理学模式方面的能力。模拟社区分析表明，引物和DNA分离的偏见和高估的多样性，减少后，消除单操作分类单位（OTU）。唾液和粘膜群落的测序发现总共455个OTU（0.3%差异），其中仅78个存在于所有受试者中。我们证明，这种变异性部分是不完整的丰富度覆盖的结果，即使在很大的测序深度，因此比较社区的结构是更有效的比较比仅基于成员资格。关于口腔粘膜造影，我们发现受试者间的社区结构的差异低于受试者内唾液和粘膜社区之间的差异。这些差异在非常低的测序深度下是明显的，并且主要是由粘膜中的缓症链球菌和溶血双生菌的丰度引起的。总之，我们提出了一个实验和数据分析框架，这将有助于设计和解释基于焦磷酸测序的研究。尽管与此相关的技术的挑战，我们证明了它的权力，口腔多样性和pneumeography模式的评估。
High throughput sequencing of 16S ribosomal RNA gene amplicons is a cost-effective method for characterization of oral bacterial communities. However, before undertaking large-scale studies, it is necessary to understand the technique-associated limitations and intrinsic variability of the oral ecosystem. In this work we evaluated bias in species representation using an in vitro-assembled mock community of oral bacteria. We then characterized the bacterial communities in saliva and buccal mucosa of five healthy subjects to investigate the power of high throughput sequencing in revealing their diversity and biogeography patterns. Mock community analysis showed primer and DNA isolation biases and an overestimation of diversity that was reduced after eliminating singleton operational taxonomic units (OTUs). Sequencing of salivary and mucosal communities found a total of 455 OTUs (0.3% dissimilarity) with only 78 of these present in all subjects. We demonstrate that this variability was partly the result of incomplete richness coverage even at great sequencing depths, and so comparing communities by their structure was more effective than comparisons based solely on membership. With respect to oral biogeography, we found inter-subject variability in community structure was lower than site differences between salivary and mucosal communities within subjects. These differences were evident at very low sequencing depths and were mostly caused by the abundance of Streptococcus mitis and Gemella haemolysans in mucosa. In summary, we present an experimental and data analysis framework that will facilitate design and interpretation of pyrosequencing-based studies. Despite challenges associated with this technique, we demonstrate its power for evaluation of oral diversity and biogeography patterns.