Department of Energy Joint Genome Institute, Walnut Creek, CA, USA.
ISME J. 2010 May;4(5):642-7. doi: 10.1038/ismej.2009.153. Epub 2010 Jan 21.
Pyrosequencing of 16S rRNA gene amplicons for microbial community profiling can, for equivalent costs, yield more than two orders of magnitude more sensitivity than traditional PCR cloning and Sanger sequencing. With this increased sensitivity and the ability to analyze multiple samples in parallel, it has become possible to evaluate several technical aspects of PCR-based community structure profiling methods. We tested the effect of amplicon length and primer pair on estimates of species richness (number of species) and evenness (relative abundance of species) by assessing the potentially tractable microbial community residing in the termite hindgut. Two regions of the 16S rRNA gene were sequenced from one of two common priming sites, spanning the V1-V2 or V8 regions, using amplicons ranging in length from 352 to 1443 bp. Our results show that both amplicon length and primer pair markedly influence estimates of richness and evenness. However, estimates of species evenness are consistent among different primer pairs targeting the same region. These results highlight the importance of experimental methodology when comparing diversity estimates across communities.
16S rRNA 基因扩增子的焦磷酸测序可用于微生物群落分析，其成本相当，但灵敏度比传统的 PCR 克隆和 Sanger 测序高出两个数量级以上。这种更高的灵敏度和同时分析多个样本的能力使得评估基于 PCR 的群落结构分析方法的几个技术方面成为可能。我们通过评估居住在白蚁后肠中的潜在可处理微生物群落，测试了扩增子长度和引物对在估计物种丰富度（物种数量）和均匀度（物种相对丰度）方面的影响。我们使用长度为 352 至 1443 bp 的扩增子，从两个常见的引物结合位点之一（V1-V2 或 V8 区）对 16S rRNA 基因的两个区域进行测序。我们的结果表明，扩增子长度和引物对都显著影响丰富度和均匀度的估计。然而，针对同一区域的不同引物对的物种均匀度估计值是一致的。这些结果强调了在比较不同群落的多样性估计值时实验方法学的重要性。
