PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets.
PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets.
复制标题
PCR在焦磷酸测序数据集中偏离细菌和古细菌群落结构。
DOI：
10.1371/journal.pone.0043093
复制
发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Raskin L
中科院分区：
文献类型：
--
作者：
Pinto AJ;Raskin L
由于16 S rRNA基因靶向大规模平行测序已成为微生物多样性研究的常用工具，因此已取得许多进展，以通过严格的质量控制措施最大限度地减少测序和嵌合PCR伪影的影响。然而，一直很少有人努力了解多模板PCR偏差对微生物群落结构的影响。在这项研究中，我们使用了三个细菌和三个古细菌模拟社区，分别由33个细菌和24个古细菌16 S rRNA基因序列以不同比例组合组成，以比较（1）测序深度，（2）测序伪影（测序错误和嵌合PCR伪影），和（3）多模板PCR中的偏差，对焦磷酸测序数据集的社区结构的解释。我们还评估了这三个变量中的每一个对α-和β-多样性度量的影响，所述α-和β-多样性度量仅依赖于OTU的数量（丰富度）以及包括成员资格和检测到的OTU的相对丰度（多样性）的那些。作为这项研究的一部分，我们重新设计了细菌和古细菌引物组，靶向16 S rRNA基因的V3-V5区域，沿着多重条形码，以允许同时测序来自两个域的PCR产物。我们得出结论，更深入的测序工作的益处超出了更高的OTU检测，并通过减少重复文库之间的变异性来提高β多样性分析的精度，尽管存在更多的测序伪影。另外，由测序错误产生的假OTU对基于丰富度或共享丰富度的α-和β-多样性度量具有显著影响，而利用群落结构（包括OTU的丰富度和相对丰度两者）的度量受假OTU的影响最小。然而，准确评估群落结构的最大障碍是由于与多模板PCR反应相关的偏差而导致的每个检测到的OTU的估计平均相对丰度的误差。
As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3–V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions.