Testing the Limits of 454 Pyrotag Sequencing: Reproducibility, Quantitative Assessment and Comparison to T-RFLP Fingerprinting of Aquifer Microbes

The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ∼4% read abundance across biological, and ∼2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific ‘in vivo’ fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities.

近年来，随着扩增子焦磷酸测序（amplicon pyrosequencing）技术的引入，依托16S rRNA基因（16S rRNA gene）谱分析表征微生物群落结构的研究获得了长足进展。通过样本条形码标记技术，我们得以大规模筛查环境或临床来源的多样本，以获取其群落细节信息。然而，当前学界仍存在持续争论，对pyrotag测序（pyrotag sequencing）的可重复性与半定量严谨性提出质疑，这与群落指纹图谱（community fingerprinting）发展初期的境遇相似。本研究以含水层沉积物中的细菌群落为研究对象，设置生物重复与技术重复样本，验证了细菌454 pyrotag测序（454 pyrotag sequencing）的可重复性。此外，本研究通过向环境DNA（environmental DNA）中添加定量的外源性模板，探究了恢复特定模板比例的可行性。我们对取自德国杜塞尔多夫一处焦油污染含水层的年度采样活动中获取的三重重复沉积物样本的pyrotag文库（pyrotag libraries）进行了测序。结果显示，优势类群的读长（read）丰度具有极高的可重复性：生物重复样本间的读长丰度最大标准差约为4%，技术重复样本间则约为2%。本研究的分析流程还可将已组装的特定pyrotag重叠群（pyrotag contigs）内的读长丰度，与特定‘体内（in vivo）’指纹特征的丰度进行关联。由此可见，末端限制性片段长度多态性（terminal restriction fragment length polymorphism, T-RFLP）分析与pyrotag测序（pyrotag sequencing）均能够获取高度一致的群落结构信息。扩增子测序（amplicon sequencing）所检测到的整体群落多样性约为其他技术的两倍。pyrotag文库（pyrotag libraries）还能够线性恢复向沉积物DNA中加标的费氏弧菌（Aliivibrio fisheri）纯培养物的16S rRNA基因（16S rRNA gene）添加比例（最高可达20%）。本研究证明，454 pyrotag测序是一种稳健且可重复的方法，能够可靠地恢复自然微生物群落中的模板丰度与整体群落结构。