An accurate and efficient experimental approach for characterization of the complex oral microbiota.
An accurate and efficient experimental approach for characterization of the complex oral microbiota.
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DOI：
10.1186/s40168-015-0110-9
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发表时间：
2015-10-05
期刊：
影响因子：
15.5
通讯作者：
Buck MJ
中科院分区：
文献类型：
--
作者：
Zheng W;Tsompana M;Ruscitto A;Sharma A;Genco R;Sun Y;Buck MJ
目前，微生物群的分类询问是基于临床和科学环境中16 S rRNA基因序列的扩增。微生物群的准确评估在很大程度上取决于所使用的引物，并且非代表性基因组区域的扩增可能会产生属/种分辨率偏差。最新的Illumina MiSeq测序化学将读取长度扩展到300 bp，从而能够以一小部分成本在单个配对末端反应中对大量样品进行深度分析。越来越多的研究人员采用这种技术进行针对16 S rRNA V3-V4高变区的各种微生物组研究。为了扩展这个强大平台在进一步描述性和功能性微生物组研究中的适用性，我们使用新的2 × 300 MiSeq平台标准化并测试了一种高效，可靠和简单的工作流程，用于16 S V1-V3高变区的扩增，文库构建和测序。我们的分析涉及11个龈下菌斑样本从糖尿病和非糖尿病人类受试者患牙周炎。将我们的实验方案的效率和可靠性与来自相同样品的16 S V3-V4测序数据进行比较。比较的基础上观察到的分类丰富度和物种均匀度的措施，沿着与Procrustes分析使用β（β）多样性距离度量。作为实验对照，我们还分析了来自具有已知细菌物种操纵子计数的合成群落的V1-V3和V3-V4区域的总共8个技术重复。我们表明，我们的实验方案准确地衡量真正的细菌群落组成。基于未加权UniFrac β多样性度量的Procrustes分析描绘了V1-V3和V3-V4区域的口腔细菌组成之间的显著相关性。然而，V1-V3区域的微生物类型丰富度较高，这表明V1-V3为复杂的口腔微生物群提供了更深入的种群多样性和群落生态评估。这项研究为研究人员选择合适的16 S扩增子用于未来的人类口腔微生物组研究提供了有价值的实验证据。我们预计，测试的16 S V1-V3框架将广泛适用于其他类型的微生物群，允许对数千个样本进行稳健、省时且廉价的检查，以用于群体、系统发育和功能横截面和潮汐研究。
Currently, taxonomic interrogation of microbiota is based on amplification of 16S rRNA gene sequences in clinical and scientific settings. Accurate evaluation of the microbiota depends heavily on the primers used, and genus/species resolution bias can arise with amplification of non-representative genomic regions. The latest Illumina MiSeq sequencing chemistry has extended the read length to 300 bp, enabling deep profiling of large number of samples in a single paired-end reaction at a fraction of the cost. An increasingly large number of researchers have adopted this technology for various microbiome studies targeting the 16S rRNA V3–V4 hypervariable region. To expand the applicability of this powerful platform for further descriptive and functional microbiome studies, we standardized and tested an efficient, reliable, and straightforward workflow for the amplification, library construction, and sequencing of the 16S V1–V3 hypervariable region using the new 2 × 300 MiSeq platform. Our analysis involved 11 subgingival plaque samples from diabetic and non-diabetic human subjects suffering from periodontitis. The efficiency and reliability of our experimental protocol was compared to 16S V3–V4 sequencing data from the same samples. Comparisons were based on measures of observed taxonomic richness and species evenness, along with Procrustes analyses using beta(β)-diversity distance metrics. As an experimental control, we also analyzed a total of eight technical replicates for the V1–V3 and V3–V4 regions from a synthetic community with known bacterial species operon counts. We show that our experimental protocol accurately measures true bacterial community composition. Procrustes analyses based on unweighted UniFrac β-diversity metrics depicted significant correlation between oral bacterial composition for the V1–V3 and V3–V4 regions. However, measures of phylotype richness were higher for the V1–V3 region, suggesting that V1–V3 offers a deeper assessment of population diversity and community ecology for the complex oral microbiota. This study provides researchers with valuable experimental evidence for the selection of appropriate 16S amplicons for future human oral microbiome studies. We expect that the tested 16S V1–V3 framework will be widely applicable to other types of microbiota, allowing robust, time-efficient, and inexpensive examination of thousands of samples for population, phylogenetic, and functional crossectional and longitutidal studies.