Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, Colorado, United States of America.
PLoS One. 2012;7(9):e44563. doi: 10.1371/journal.pone.0044563. Epub 2012 Sep 4.
Since the composition of the human microbiome is highly variable both within and between individuals, researchers are increasingly reliant on high-throughput molecular approaches to identify linkages between the composition of these communities and human health. While new sequencing technologies have made it increasingly feasible to analyze large numbers of human-associated samples, the extraction of DNA from samples often remains a bottleneck in the process. Here we tested a direct PCR approach using the Extract-N-Amp Plant PCR Kit to accelerate the 16S rRNA gene-based analyses of human-associated bacterial communities, directly comparing this method to a more commonly-used approach whereby DNA is first extracted and purified from samples using a series of steps prior to PCR amplification. We used both approaches on replicate samples collected from each of five body habitats (tongue surface, feces, forehead skin, underarm skin, and forearm skin) from four individuals. With the exception of the tongue samples, there were few significant differences in the estimates of taxon richness or phylogenetic diversity obtained using the two approaches. Perhaps more importantly, there were no significant differences between the methods in their ability resolve body habitat differences or inter-individual differences in bacterial community composition and the estimates of the relative abundances of individual taxa were nearly identical with the two methods. Overall, the two methods gave very similar results and the direct PCR approach is clearly advantageous for many studies exploring the diversity and composition of human-associated bacterial communities given that large numbers of samples can be processed far more quickly and efficiently.
由于人类微生物组的组成在个体内部和个体之间高度可变,研究人员越来越依赖高通量分子方法来确定这些群落的组成与人类健康之间的联系。虽然新的测序技术使得分析大量与人类相关的样本变得越来越可行,但从样本中提取 DNA 仍然是该过程中的一个瓶颈。在这里,我们使用 Extract-N-Amp 植物 PCR 试剂盒测试了一种直接 PCR 方法,以加速基于 16S rRNA 基因的人类相关细菌群落的分析,直接将该方法与更常用的方法进行比较,即在 PCR 扩增之前,先通过一系列步骤从样本中提取和纯化 DNA。我们在来自四个人的五个身体栖息地(舌表面、粪便、额部皮肤、腋窝皮肤和前臂皮肤)的重复样本上使用了这两种方法。除了舌样本外,两种方法获得的分类丰富度或系统发育多样性估计值很少有显著差异。也许更重要的是,这两种方法在解析身体栖息地差异或个体间细菌群落组成差异方面的能力没有显著差异,并且两种方法对个体分类群相对丰度的估计值几乎相同。总体而言,这两种方法产生了非常相似的结果,对于许多探索人类相关细菌群落多样性和组成的研究来说,直接 PCR 方法显然具有优势,因为可以更快速、更有效地处理大量样本。