Environmental Biotechnology Laboratory, Department of Civil Engineering, The University of Hong Kong, Hong Kong SAR, China.
PLoS One. 2013;8(1):e53649. doi: 10.1371/journal.pone.0053649. Epub 2013 Jan 14.
In recent years, PCR-based pyrosequencing of 16S rRNA genes has continuously increased our understanding of complex microbial communities in various environments of the Earth. However, there is always concern on the potential biases of diversity determination using different 16S rRNA gene primer sets and covered regions. Here, we first report how bacterial 16S rRNA gene pyrotags derived from a series of different primer sets resulted in the biased diversity metrics. In total, 14 types of pyrotags were obtained from two-end pyrosequencing of 7 amplicon pools generated by 7 primer sets paired by 1 of 4 forward primers (V1F, V3F, V5F, and V7F) and 1 of 4 reverse primers (V2R, V4R, V6R, and V9R), respectively. The results revealed that: i) the activated sludge exhibited a large bacterial diversity that represented a broad range of bacterial populations and served as a good sample in this methodology research; ii) diversity metrics highly depended on the selected primer sets and covered regions; iii) paired pyrotags obtained from two-end pyrosequencing of each short amplicon displayed different diversity metrics; iv) relative abundance analysis indicated the sequencing depth affected the determination of rare bacteria but not abundant bacteria; v) the primer set of V1F and V2R significantly underestimated the diversity of activated sludge; and vi) the primer set of V3F and V4R was highly recommended for future studies due to its advantages over other primer sets. All of these findings highlight the significance of this methodology research and offer a valuable reference for peer researchers working on microbial diversity determination.
近年来，基于 16S rRNA 基因的 PCR 焦磷酸测序技术不断提高了我们对地球各种环境中复杂微生物群落的认识。然而，使用不同的 16S rRNA 基因引物和覆盖区域来确定多样性时，始终存在潜在的偏差。在这里，我们首先报告了不同的 16S rRNA 基因引物系列产生的细菌 16S rRNA 基因焦磷酸标签如何导致多样性指标出现偏差。总共从 7 个引物组的双端焦磷酸测序中获得了 14 种焦磷酸标签，这 7 个引物组由 4 个正向引物（V1F、V3F、V5F 和 V7F）之一和 4 个反向引物（V2R、V4R、V6R 和 V9R）之一配对组成。结果表明：i）活性污泥表现出较大的细菌多样性，代表了广泛的细菌种群，是本方法研究的良好样本；ii）多样性指标高度依赖于所选的引物和覆盖区域；iii）从每个短扩增子的双端焦磷酸测序获得的配对焦磷酸标签显示出不同的多样性指标；iv）相对丰度分析表明测序深度影响稀有细菌的确定，但不影响丰富细菌的确定；v）V1F 和 V2R 引物组显著低估了活性污泥的多样性；vi）V3F 和 V4R 引物组由于其优于其他引物组的优势，因此强烈推荐用于未来的研究。所有这些发现突出了这种方法研究的重要性，并为从事微生物多样性测定的同行研究人员提供了有价值的参考。
