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We present an evolutionary placement algorithm (EPA) and a Web server for the rapid assignment of sequence fragments (short reads) to edges of a given phylogenetic tree under the maximum-likelihood model. The accuracy of the algorithm is evaluated on several real-world data sets and compared with placement by pair-wise sequence comparison, using edit distances and BLAST. We introduce a slow and accurate as well as a fast and less accurate placement algorithm. For the slow algorithm, we develop additional heuristic techniques that yield almost the same run times as the fast version with only a small loss of accuracy. When those additional heuristics are employed, the run time of the more accurate algorithm is comparable with that of a simple BLAST search for data sets with a high number of short query sequences. Moreover, the accuracy of the EPA is significantly higher, in particular when the sample of taxa in the reference topology is sparse or inadequate. Our algorithm, which has been integrated into RAxML, therefore provides an equally fast but more accurate alternative to BLAST for tree-based inference of the evolutionary origin and composition of short sequence reads. We are also actively developing a Web server that offers a freely available service for computing read placements on trees using the EPA.

Background Shotgun sequencing of environmental DNA is an essential technique for characterizing uncultivated microbes in situ . However, the taxonomic and functional assignment of the obtained sequence fragments remains a pressing problem. Results Existing algorithms are largely optimized for speed and coverage; in contrast, we present here a software framework that focuses on a restricted set of informative gene families, using Maximum Likelihood to assign these with the best possible accuracy. This framework ('MLTreeMap'; http://mltreemap.org/ ) uses raw nucleotide sequences as input, and includes hand-curated, extensible reference information. Conclusions We discuss how we validated our pipeline using complete genomes as well as simulated and actual environmental sequences.

Background Aligning short DNA reads to a reference sequence alignment is a prerequisite for detecting their biological origin and analyzing them in a phylogenetic context. With the PaPaRa tool we introduced a dedicated dynamic programming algorithm for simultaneously aligning short reads to reference alignments and corresponding evolutionary reference trees. The algorithm aligns short reads to phylogenetic profiles that correspond to the branches of such a reference tree. The algorithm needs to perform an immense number of pairwise alignments. Therefore, we explore vector intrinsics and GPUs to accelerate the PaPaRa alignment kernel. Results We optimized and parallelized PaPaRa on CPUs and GPUs. Via SSE 4.1 SIMD (Single Instruction, Multiple Data) intrinsics for x86 SIMD architectures and multi-threading, we obtained a 9-fold acceleration on a single core as well as linear speedups with respect to the number of cores. The peak CPU performance amounts to 18.1 GCUPS (Giga Cell Updates per Second) using all four physical cores on an Intel i7 2600 CPU running at 3.4 GHz. The average CPU performance (averaged over all test runs) is 12.33 GCUPS. We also used OpenCL to execute PaPaRa on a GPU SIMT (Single Instruction, Multiple Threads) architecture. A NVIDIA GeForce 560 GPU delivered peak and average performance of 22.1 and 18.4 GCUPS respectively. Finally, we combined the SIMD and SIMT implementations into a hybrid CPU-GPU system that achieved an accumulated peak performance of 33.8 GCUPS. Conclusions This accelerated version of PaPaRa (available at http://www.exelixis-lab.org/software.html ) provides a significant performance improvement that allows for analyzing larger datasets in less time. We observe that state-of-the-art SIMD and SIMT architectures deliver comparable performance for this dynamic programming kernel when the “competing programmer approach” is deployed. Finally, we show that overall performance can be substantially increased by designing a hybrid CPU-GPU system with appropriate load distribution mechanisms.

Nucleotide positions in the hypervariable V4 and V9 regions of the small subunit (SSU)-rDNA locus are normally difficult to align and are usually removed before standard phylogenetic analyses. Yet, with next-generation sequencing data, amplicons of these regions are all that are available to answer ecological and evolutionary questions that rely on phylogenetic inferences. With ciliates, we asked how inclusion of the V4 or V9 regions, regardless of alignment quality, affects tree topologies using distinct phylogenetic methods (including PairDist that is introduced here). Results show that the best approach is to place V4 amplicons into an alignment of full-length Sanger SSU-rDNA sequences and to infer the phylogenetic tree with RAxML. A sliding window algorithm as implemented in RAxML shows, though, that not all nucleotide positions in the V4 region are better than V9 at inferring the ciliate tree. With this approach and an ancestral-state reconstruction, we use V4 amplicons from European nearshore sampling sites to infer that rather than being primarily terrestrial and freshwater, colpodean ciliates may have repeatedly transitioned from terrestrial/freshwater to marine environments.

The internal transcribed spacer region (ITS) of the nuclear rDNA cistron represents the barcoding locus for Fungi. Intragenomic variation of this multicopy gene can interfere with accurate phylogenetic reconstruction of biological entities. We investigated the amount and nature of this variation for the lichenized fungus Cora inversa in the Hygrophoraceae (Basidiomycota: Agaricales), analyzing base call and length variation in ITS1 454 pyrosequencing data of three samples of the target mycobiont, for a total of 16,665 reads obtained from three separate repeats of the same samples under different conditions. Using multiple fixed alignment methods (PaPaRa) and maximum likelihood phylogenetic analysis (RAxML), we assessed phylogenetic relationships of the obtained reads, together with Sanger ITS sequences from the same samples. Phylogenetic analysis showed that all ITS1 reads belonged to a single species, C. inversa . Pyrosequencing data showed 266 insertion sites in addition to the 325 sites expected from Sanger sequences, for a total of 15,654 insertions (0.94 insertions per read). An additional 3,279 substitutions relative to the Sanger sequences were detected in the dataset, out of 5,461,125 bases to be called. Up to 99.3 % of the observed indels in the dataset could be interpreted as 454 pyrosequencing errors, approximately 65 % corresponding to incorrectly recovered homopolymer segments, and 35 % to carry-forward-incomplete-extension errors. Comparison of automated clustering and alignment-based phylogenetic analysis demonstrated that clustering of these reads produced a 35-fold overestimation of biological diversity in the dataset at the 95 % similarity threshold level, whereas phylogenetic analysis using a maximum likelihood approach accurately recovered a single biological entity. We conclude that variation detected in 454 pyrosequencing data must be interpreted with great care and that a combination of a sufficiently large number of reads per taxon, a set of Sanger references for the same taxon, and at least two runs under different emulsion PCR and sequencing conditions, are necessary to reliably separate biological variation from 454 sequencing errors. Our study shows that clustering methods are highly sensitive to artifactual sequence variation and inadequate to properly recover biological diversity in a dataset, if sequencing errors are substantial and not removed prior to clustering analysis.

To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed that on average 43% of the species abundance and 58% of the richness cannot be captured by current reference genome-based methods. An implementation of the method is available at http://www.bork.embl.de/software/mOTU/.

Despite their potential shortcomings for phylogenetic inference, sometimes only morphological data are available for systematic classification. Computational methods are used to determine morphological site patterns congruent with the molecular tree in order to improve the accuracy of the classification of taxa for which only morphological data exist. Here, we used molecular site weight calibration as implemented in RA×ML to weight morphological characters based on their distribution on a maximum likelihood tree inferred from molecular data. We subsequently conducted morphology-based 'phylogenetic binning' (= assignment of morphologically defined taxa that lack molecular data to branches of the molecular reference tree) based on the morphology of the taxa included in the tree. We applied this methodology to the lichen genus Graphis s.l. (Ascomycota: Ostropales: Graphidaceae), which was recently shown to represent two separate, distantly related lineages, Graphis s.str. and Allographa. Of 313 species of Graphis and Allographa included in the study, 16 were represented by molecular sequence data and morphology, and 297 by morphology only. Using maximum likelihood and maximum parsimony site weight calibration and morphology-based phylogenetic binning, 281-290 of the 297 species represented by morphological data only could be assigned to either Graphis or Allographa with strong support (90%-100%). Two species, G. saxiseda and G. evirescens, were found to represent species of the unrelated genus Carbacanthographis. Our results showed that assignment of taxa to clades based on morphological data only substantially improved with molecular site weight calibration. Both molecular site weight calibration and branch assignment to the molecular reference tree are implemented in the RA×ML v. 7.2.6 Windows executable and the RA×ML v. 7.2.8 open-source code.

We develop, estimate, and test a tractable general equilibrium model of oligopsony with differentiated jobs and concentrated labor markets. We estimate key model parameters by matching new evidence on the relationship between firms’ local labor market share and their employment and wage responses to state corporate tax changes. The model quantitatively replicates quasi-experimental evidence on imperfect productivity-wage pass-through and strategic wage setting of dominant employers. Relative to the efficient allocation, welfare losses from labor market power are 7.6 percent, while output is 20.9 percent lower. Lastly, declining local concentration added 4 percentage points to labor’s share of income between 1977 and 2013.

The controlled functionalization of single-walled carbon nanotubes with luminescent sp 3 -defects has created the potential to employ them as quantum-light sources in the near-infrared. For that, it is crucial to control their spectral diversity. The emission wavelength is determined by the binding configuration of the defects rather than the molecular structure of the attached groups. However, current functionalization methods produce a variety of binding configurations and thus emission wavelengths. We introduce a simple reaction protocol for the creation of only one type of luminescent defect in polymer-sorted (6,5) nanotubes, which is more red-shifted and exhibits longer photoluminescence lifetimes than the commonly obtained binding configurations. We demonstrate single-photon emission at room temperature and expand this functionalization to other polymer-wrapped nanotubes with emission further in the near-infrared. As the selectivity of the reaction with various aniline derivatives depends on the presence of an organic base we propose nucleophilic addition as the reaction mechanism. Covalent functionalization of single-walled carbon nanotubes with luminescent sp 3 -defects generally produces a variety of binding configurations and emission wavelengths. The authors propose a base-mediated nucleophilic functionalization approach to selectively achieve configurations for E 11 * and E 11 * - or purely E 11 * - defect emission.

The sumoylation (SUMO) pathway is involved in a variety of processes during C . elegans development, such as gonadal and vulval fate specification, cell cycle progression and maintenance of chromosome structure. The ubiquitous expression and pleiotropic effects have made it difficult to dissect the tissue-specific functions of the SUMO pathway and identify its target proteins. To overcome these challenges, we have established tools to block protein sumoylation and degrade sumoylated target proteins in a tissue-specific and temporally controlled manner. We employed the auxin-inducible protein degradation system (AID) to down-regulate the SUMO E3 ligase GEI-17 or the SUMO ortholog SMO-1, either in the vulval precursor cells (VPCs) or in the gonadal anchor cell (AC). Our results indicate that the SUMO pathway acts in multiple tissues to control different aspects of vulval development, such as AC positioning, basement membrane (BM) breaching, VPC fate specification and morphogenesis. Inhibition of protein sumoylation in the VPCs resulted in abnormal toroid formation and ectopic cell fusions during vulval morphogenesis. In particular, sumoylation of the ETS transcription factor LIN-1 at K169 is necessary for the proper contraction of the ventral vulA toroids. Thus, the SUMO pathway plays several distinct roles throughout vulval development.