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-cell PCR followed by Sanger sequencing to provide an unambiguous link to the NGS data. We show that NGS and morphological analyses generally capture frequency shifts of abundant taxa over our seasonal samples. The observed incongruencies are probably largely due to rDNA copy number variation among taxa and heterogeneity in the efficiency of cell lysis. Overall, NGS-based amplicon sequencing was superior in detecting rare species. We propose that in the 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.

With the advent of molecular methods, it became clear that microbial biodiversity had been vastly underestimated. Since then, species abundance patterns were determined for several environments, but temporal changes in species composition were not studied to the same level of resolution. Using massively parallel sequencing on the 454 GS FLX platform we identified a highly dynamic turnover of the seasonal abundance of protists in the Austrian lake Fuschlsee. We show that seasonal abundance patterns of protists closely match their biogeographic distribution. The stable predominance of few highly abundant taxa, which previously led to the suggestion of a low global protist species richness, is contrasted by a highly dynamic turnover of rare species. We suggest that differential seasonality of rare and abundant protist taxa explains the—so far—conflicting evidence in the ‘everything is everywhere’ dispute. Consequently temporal sampling is basic for adequate diversity and species richness estimates.

Massively parallel sequencing holds great promise for expression profiling, as it combines the high throughput of SAGE with the accuracy of EST sequencing. Nevertheless, until now only very limited information had been available on the suitability of the current technology to meet the requirements. Here, we evaluate the potential of 454 sequencing technology for expression profiling using Drosophila melanogaster. We show that short (< ∼80 bp) and long (> ∼300-400 bp) cDNA fragments are under-represented in 454 sequence reads. Nevertheless, sequencing of 3Ј cDNA fragments generated by nebulization could be used to overcome the length bias of the 454 sequencing technology. Gene expression measurements generated by restriction analysis and nebulization for fragments within the 80-to 300-bp range showed correlations similar to those reported for replicated microarray experiments (0.83-0.91); 97% of the cDNA fragments could be unambiguously mapped to the genomic DNA, demonstrating the advantage of longer sequence reads. Our analyses suggest that the 454 technology has a large potential for expression profiling, and the high mapping accuracy indicates that it should be possible to compare expression profiles across species.[Supplemental material is available online at www.genome.org. The EST sequences have been deposited in GenBank under accession nos. EV574767-EV600806.]Gene expression technologies have greatly matured over the past years, but it has become clear that hybridization-based approaches have obvious limitations in cross-species comparisons (Gilad et al. 2005(Gilad et al. , 2006. Probably the most eminent problems are mismatches in heterologous probes and probe-specific hybridization kinetics, which complicate the design of speciesspecific oligonucleotide arrays. Alternatively, sequencing-based approaches could be used to measure gene expression if the sequence reads could be unambiguously mapped to the corresponding transcripts. While the short sequence reads of serial analysis of gene expression (SAGE) (Velculescu et al. 1995) and related techniques are severely limited by the requirement of a reliable genome annotation, the recently developed 454 sequencing technology (Margulies et al. 2005) may provide sufficient sequence information to overcome this limitation at moderate costs.In this study, we evaluate the potential of 454 sequencing technology to serve as a reliable tool for expression profiling. We show that 454 sequencing technology has a biased representation of cDNA fragments with different length. However, in combination with random breakage of the cDNAs by nebulization, 454 sequencing provides an excellent tool for expression profiling. The high accuracy with which we could map the sequenced fragments onto the Drosophila melanogaster genome suggests that 454 sequencing has great potential for interspecific expression profiling. Results Conceptual designMeasuring gene expression by sequencing requires only that a proportion of the transcript be analyzed. We sequenced a 3Ј region of the cDNA to...