18S rRNA sequencing of 10 seasonal eukaryotic freshwater samples on 454 GS FLX

With the delivery of millions of sequence reads in a single experiment, next generation sequencing (NGS) is currently revolutionizing surveys of microorganism diversity. In particular, when applied to Eukaryotes, we are still lacking a rigorous comparison of morphological and NGS based diversity estimates. In this report, we studied the diversity and the seasonal community turnover of alveolates (Ciliophora and Dinophyceae) in an oligotrophic freshwater lake by SSU amplicon sequencing with NGS as well as by classical morphological analysis. We complemented the morphological analysis by single molecule PCR followed by Sanger sequencing to provide an unambiguous link to the NGS data. We show that NGS sequencing of rDNA does not always adequately reflect the species abundance, probably due to rDNA copy number variation among taxa and heterogeneity in the efficiency of cell lysis. Overall, NGS and morphological analyses capture frequency shifts of abundant taxa over our seasonal samples. NGS based amplicon sequencing was superior in detecting rare species. We propose that in absence of other nuclear markers less susceptible to copy number variation, rDNA-based diversity studies need to be adjusted for confounding effects of copy number variation.