Abstract
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.
Highlights
Since the introduction of generation sequencing technologies, a rapid growth of applications and developments in the screening of complex microbial communities has been observed [1,2]
We systematically investigate the reproducibility of this pyrotag sequencing approach across biological and technical replicates, we compare the recovery of relative operational taxonomic unit (OTU) abundances between pyrotag libraries and terminal restriction fragment length polymorphism (T-RFLP) and, by spiking of environmental DNA extracts with qPCR defined template amendments, we explore the potential of the method for recovering input ratios of specific taxa within complex microbial communities
The reproducibility of pyrotag sequencing in our workflow was generally high, with phylum/class-level read abundances across biological replicates retrieved at an average standard deviation (SD) of,0.6% (Figure 1A, maximum SD of 4.3%)
Summary
Since the introduction of generation sequencing technologies, a rapid growth of applications and developments in the screening of complex microbial communities has been observed [1,2]. The general reproducibility and robustness of pyrotag sequencing, its potential to adequately recover relative template abundances, as well as its comparability to other, more established microbial community screening techniques like rRNA gene fingerprinting are still a matter of debate.
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