Back to basics – the influence of DNA extraction and primer choice on phylogenetic analysis in activated sludge communities

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DNA extraction and primer choice have a large effect on the observed community structure in all phylogenetic analyses. Although the biases are well known, no comprehensive analysis have been conducted in activated sludge communities. In this study we investigated the effect of bead beating intensity and primer choice on the observed community using 16S rDNA amplicon sequencing. Quantitative fluorescence in situ hybridization (qFISH) was used as a DNA extraction independent method to evaluate the results. The bead beating intensity correlated with cell-wall strength and showed that the manufacture recommended settings were insufficient to retrieve a large part of the community. In addition, the in silico “best” primer set was found to greatly underestimate a number of important phyla when compared to qFISH results. The findings underline the need for sample specific and DNA extraction independent validation in all DNA extraction based studies.
StatusUdgivet - 2013
BegivenhedMicrobial Ecology and Water Engineering 2013 - Ann Arbor, Michigan, USA
Varighed: 7 jul. 201310 jul. 2013
Konferencens nummer: 5


KonferenceMicrobial Ecology and Water Engineering 2013
ByAnn Arbor, Michigan