testing the limits of 454 pyrotag sequencing reproducibility, quantitative assessment and comparison to t-rflp fingerprinting of aquifer microbes测试454的极限pyrotag测序再现性,定量评估和比较t-rflp指纹识别含水层的微生物.pdfVIP

Testing the Limits of 454 Pyrotag Sequencing: Reproducibility, Quantitative Assessment and Comparison to T-RFLP Fingerprinting of Aquifer Microbes 1 1 2 1 Giovanni Pilloni , Michael S. Granitsiotis , Marion Engel , Tillmann Lueders * ¨ 1 Institute of Groundwater Ecology, Helmholtz Zentrum Munchen - German Research Centre for Environmental Health, Neuherberg, Germany, 2 Research Unit ¨ Environmental Genomics, Helmholtz Zentrum Munchen - German Research Centre for Environmental Health, Neuherberg, Germany 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 Dusseldorf, Germany. The abundance of dominatin