Summary: Sequencing pooled DNA samples (Pool-Seq) is the most cost-effective approach for the genome-wide comparison of population samples. Here, we introduce PoPoolation2, the first software tool specifically designed for the comparison of populations with Pool-Seq data. PoPoolation2 implements a range of commonly used measures of differentiation (FST, Fisher's exact test and Cochran-Mantel-Haenszel test) that can be applied on different scales (windows, genes, exons, SNPs). The result may be visualized with the widely used Integrated Genomics Viewer.Availability and Implementation: PoPoolation2 is implemented in Perl and R. It is freely available on http://code.google.com/p/popoolation2/Contact: christian.schloetterer@vetmeduni.ac.atSupplementary Information: Manual: http://code.google.com/p/popoolation2/wiki/Manual Test data and tutorial: http://code.google.com/p/popoolation2/wiki/Tutorial Validation: http://code.google.com/p/popoolation2/wiki/Validation

Recent statistical analyses suggest that sequencing of pooled samples provides a cost effective approach to determine genome-wide population genetic parameters. Here we introduce PoPoolation, a toolbox specifically designed for the population genetic analysis of sequence data from pooled individuals. PoPoolation calculates estimates of θ Watterson, θ π, and Tajima's D that account for the bias introduced by pooling and sequencing errors, as well as divergence between species. Results of genome-wide analyses can be graphically displayed in a sliding window plot. PoPoolation is written in Perl and R and it builds on commonly used data formats. Its source code can be downloaded from http://code.google.com/p/popoolation/. Furthermore, we evaluate the influence of mapping algorithms, sequencing errors, and read coverage on the accuracy of population genetic parameter estimates from pooled data.

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.

Although it is well understood that selection shapes the polymorphism pattern in Drosophila, signatures of classic selective sweeps are scarce. Here, we focus on Drosophila mauritiana, an island endemic, which is closely related to Drosophila melanogaster. Based on a new, annotated genome sequence, we characterized the genome-wide polymorphism by sequencing pooled individuals (Pool-seq). We show that the interplay between selection and recombination results in a genome-wide polymorphism pattern characteristic for D. mauritiana. Two large genomic regions (>500 kb) showed the signature of almost complete selective sweeps. We propose that the absence of population structure and limited geographic distribution could explain why such pronounced sweep patterns are restricted to D. mauritiana. Further evidence for strong adaptive evolution was detected for several nucleoporin genes, some of which were not previously identified as genes involved in genomic conflict. Since this adaptive evolution is continuing after the split of D. mauritiana and Drosophila simulans, we conclude that genomic conflict is not restricted to short episodes, but rather an ongoing process in Drosophila.