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Plants produce diverse metabolites to cope with the challenges presented by complex and ever-changing environments. These challenges drive the diversification of specialized metabolites within and between plant species. However, we are just beginning to understand how frequently new alleles arise controlling specialized metabolite diversity and how the geographic distribution of these alleles may be structured by ecological and demographic pressures. Here, we measure the variation in specialized metabolites across a population of 797 natural Arabidopsis thaliana accessions. We show that a combination of geography, environmental parameters, demography and different genetic processes all combine to influence the specific chemotypes and their distribution. This showed that causal loci in specialized metabolism contain frequent independently generated alleles with patterns suggesting potential within-species convergence. This provides a new perspective about the complexity of the selective forces and mechanisms that shape the generation and distribution of allelic variation that may influence local adaptation., eLife digest Since plants cannot move, they have evolved chemical defenses to help them respond to changes in their surroundings. For example, where animals run from predators, plants may produce toxins to put predators off. This approach is why plants are such a rich source of drugs, poisons, dyes and other useful substances. The chemicals plants produce are known as specialized metabolites, and they can change a lot between, and even within, plant species. The variety of specialized metabolites is a result of genetic changes and evolution over millions of years. Evolution is a slow process, yet plants are able to rapidly develop new specialized metabolites to protect them from new threats. Even different populations of the same species produce many distinct metabolites that help them survive in their surroundings. However, the factors that lead plants to produce new metabolites are not well understood, and it is not known how this affects genetic variation. To gain a better understanding of this process, Katz et al. studied 797 European variants of a common weed species called Arabidopsis thaliana, which is widely studied. The investigation found that many factors affect the range of specialized metabolites in each variant. These included local geography and environment, as well as genetics and population history (demography). Katz et al. revealed a pattern of relationships between the variants that could mirror their evolutionary history as the species spread and adapted to new locations. These results highlight the complex network of factors that affect plant evolution. Rapid diversification is key to plant survival in new and changing environments and has resulted in a wide range of specialized metabolites. As such they are of interest both for studying plant evolution and for understanding their ecology. Expanding similar work to more populations and other species will broaden the scope of our ability to understand how plants adapt to their surroundings.

The presence of pathogen-specific antibodies in an individual’s blood-sample is used as an indication of previous exposure and infection to that specific pathogen (e.g., virus or bacterium). Measurement of the diagnostic antibodies is routinely achieved using solid phase immuno-assays such as ELISA tests and western blots. Here, we describe a sero-diagnostic approach based on phage-display of epitope arrays we term “Domain-Scan”. We harness Next-generation sequencing (NGS) to measure the serum binding to dozens of epitopes derived from HIV-1 and HCV simultaneously. The distinction of healthy individuals from those infected with either HIV-1 or HCV, is modeled as a machine-learning classification problem, in which each determinant (“domain”) is considered as a feature, and its NGS read-out provides values that correspond to the level of determinant-specific antibodies in the sample. We show that following training of a machine-learning model on labeled examples, we can very accurately classify unlabeled samples and pinpoint the domains that contribute most to the classification. Our experimental/computational Domain-Scan approach is general and can be adapted to other pathogens as long as sufficient training samples are provided.

Cation/proton antiporters (CPAs) play a major role in maintaining living cells’ homeostasis. CPAs are commonly divided into two main groups, CPA1 and CPA2, and are further characterized by two main phenotypes: ion selectivity and electrogenicity. However, tracing the evolutionary relationships of these transporters is challenging because of the high diversity within CPAs. Here, we conduct comprehensive evolutionary analysis of 6537 representative CPAs, describing the full complexity of their phylogeny, and revealing a sequence motif that appears to determine central phenotypic characteristics. In contrast to previous suggestions, we show that the CPA1/CPA2 division only partially correlates with electrogenicity. Our analysis further indicates two acidic residues in the binding site that carry the protons in electrogenic CPAs, and a polar residue in the unwound transmembrane helix 4 that determines ion selectivity. A rationally designed triple mutant successfully converted the electrogenic CPA, EcNhaA, to be electroneutral., Cation/proton antiporters (CPAs) play a major role in maintaining living cells’ homeostasis and are divided in two main groups: CPA1 and CPA2. Here authors use a comprehensive evolutionary analysis of 6537 representative CPAs and reveal a sequence motif that determines central phenotypic characteristics.

The most common evolutionary events at the molecular level are single-base substitutions, as well as insertions and deletions (indels) of short DNA segments. A large body of research has been devoted to develop probabilistic substitution models and to infer their parameters using likelihood and Bayesian approaches. In contrast, relatively little has been done to model indel dynamics, probably due to the difficulty in writing explicit likelihood functions. Here, we contribute to the effort of modeling indel dynamics by presenting SpartaABC, an approximate Bayesian computation (ABC) approach to infer indel parameters from sequence data (either aligned or unaligned). SpartaABC circumvents the need to use an explicit likelihood function by extracting summary statistics from simulated sequences. First, summary statistics are extracted from the input sequence data. Second, SpartaABC samples indel parameters from a prior distribution and uses them to simulate sequences. Third, it computes summary statistics from the simulated sets of sequences. By computing a distance between the summary statistics extracted from the input and each simulation, SpartaABC can provide an approximation to the posterior distribution of indel parameters as well as point estimates. We study the performance of our methodology and show that it provides accurate estimates of indel parameters in simulations. We next demonstrate the utility of SpartaABC by studying the impact of alignment errors on the inference of positive selection. A C ++ program implementing SpartaABC is freely available in http://spartaabc.tau.ac.il.

Understanding species adaptation at the molecular level has been a central goal of evolutionary biology and genomics research. This important task becomes increasingly relevant with the constant rise in both genotypic and phenotypic data availabilities. The TraitRateProp web server offers a unique perspective into this task by allowing the detection of associations between sequence evolution rate and whole-organism phenotypes. By analyzing sequences and phenotypes of extant species in the context of their phylogeny, it identifies sequence sites in a gene/protein whose evolutionary rate is associated with shifts in the phenotype. To this end, it considers alternative histories of whole-organism phenotypic changes, which result in the extant phenotypic states. Its joint likelihood framework that combines models of sequence and phenotype evolution allows testing whether an association between these processes exists. In addition to predicting sequence sites most likely to be associated with the phenotypic trait, the server can optionally integrate structural 3D information. This integration allows a visual detection of trait-associated sequence sites that are juxtapose in 3D space, thereby suggesting a common functional role. We used TraitRateProp to study the shifts in sequence evolution rate of the RPS8 protein upon transitions into heterotrophy in Orchidaceae. TraitRateProp is available at http://traitrate.tau.ac.il/prop.

Many analyses for the detection of biological phenomena rely on a multiple sequence alignment as input. The results of such analyses are often further studied through parametric bootstrap procedures, using sequence simulators. One of the problems with conducting such simulation studies is that users currently have no means to decide which insertion and deletion (indel) parameters to choose, so that the resulting sequences mimic biological data. Here, we present SpartaABC, a web server that aims to solve this issue. SpartaABC implements an approximate-Bayesian-computation rejection algorithm to infer indel parameters from sequence data. It does so by extracting summary statistics from the input. It then performs numerous sequence simulations under randomly sampled indel parameters. By computing a distance between the summary statistics extracted from the input and each simulation, SpartaABC retains only parameters behind simulations close to the real data. As output, SpartaABC provides point estimates and approximate posterior distributions of the indel parameters. In addition, SpartaABC allows simulating sequences with the inferred indel parameters. To this end, the sequence simulators, Dawg 2.0 and INDELible were integrated. Using SpartaABC we demonstrate the differences in indel dynamics among three protein-coding genes across mammalian orthologs. SpartaABC is freely available for use at http://spartaabc.tau.ac.il/webserver.

The degree of evolutionary conservation of an amino acid in a protein or a nucleic acid in DNA/RNA reflects a balance between its natural tendency to mutate and the overall need to retain the structural integrity and function of the macromolecule. The ConSurf web server (http://consurf.tau.ac.il), established over 15 years ago, analyses the evolutionary pattern of the amino/nucleic acids of the macromolecule to reveal regions that are important for structure and/or function. Starting from a query sequence or structure, the server automatically collects homologues, infers their multiple sequence alignment and reconstructs a phylogenetic tree that reflects their evolutionary relations. These data are then used, within a probabilistic framework, to estimate the evolutionary rates of each sequence position. Here we introduce several new features into ConSurf, including automatic selection of the best evolutionary model used to infer the rates, the ability to homology-model query proteins, prediction of the secondary structure of query RNA molecules from sequence, the ability to view the biological assembly of a query (in addition to the single chain), mapping of the conservation grades onto 2D RNA models and an advanced view of the phylogenetic tree that enables interactively rerunning ConSurf with the taxa of a sub-tree.

Reproducible and robust data on antibody repertoires are invaluable for basic and applied immunology. Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology, providing quantitative molecular information on antibody polyclonal composition. However, major computational challenges exist when analyzing antibody sequences, from error handling to hypermutation profiles and clonal expansion analyses. In this work, we developed the ASAP (A webserver for Immunoglobulin-Seq Analysis Pipeline) webserver (https://asap.tau.ac.il). The input to ASAP is a paired-end sequence dataset from one or more replicates, with or without unique molecular identifiers. These datasets can be derived from NGS of human or murine antibody variable regions. ASAP first filters and annotates the sequence reads using public or user-provided germline sequence information. The ASAP webserver next performs various calculations, including somatic hypermutation level, CDR3 lengths, V(D)J family assignments, and V(D)J combination distribution. These analyses are repeated for each replicate. ASAP provides additional information by analyzing the commonalities and differences between the repeats (“joint” analysis). For example, ASAP examines the shared variable regions and their frequency in each replicate to determine which sequences are less likely to be a result of a sample preparation derived and/or sequencing errors. Moreover, ASAP clusters the data to clones and reports the identity and prevalence of top ranking clones (clonal expansion analysis). ASAP further provides the distribution of synonymous and non-synonymous mutations within the V genes somatic hypermutations. Finally, ASAP provides means to process the data for proteomic analysis of serum/secreted antibodies by generating a variable region database for liquid chromatography high resolution tandem mass spectrometry (LC-MS/MS) interpretation. ASAP is user-friendly, free, and open to all users, with no login requirement. ASAP is applicable for researchers interested in basic questions related to B cell development and differentiation, as well as applied researchers who are interested in vaccine development and monoclonal antibody engineering. By virtue of its user-friendliness, ASAP opens the antibody analysis field to non-expert users who seek to boost their research with immune repertoire analysis.

Classic alignment algorithms utilize scoring functions which maximize similarity or minimize edit distances. These scoring functions account for both insertion-deletion (indel) and substitution events. In contrast, alignments based on stochastic models aim to explicitly describe the evolutionary dynamics of sequences by inferring relevant probabilistic parameters from input sequences. Despite advances in stochastic modeling during the last two decades, scoring-based methods are still dominant, partially due to slow running times of probabilistic approaches. Alignment inference using stochastic models involves estimating the probability of events, such as the insertion or deletion of a specific number of characters. In this work, we present SimBa-SAl, a simulation-based approach to statistical alignment inference, which relies on an explicit continuous time Markov model for both indels and substitutions. SimBa-SAl has several advantages. First, using simulations, it decouples the estimation of event probabilities from the inference stage, which allows the introduction of accelerations to the alignment inference procedure. Second, it is general and can accommodate various stochastic models of indel formation. Finally, it allows computing the maximum-likelihood alignment, the probability of a given pair of sequences integrated over all possible alignments, and sampling alternative alignments according to their probability. We first show that SimBa-SAl allows accurate estimation of parameters of the long-indel model previously developed by Miklos et al. (2004). We next show that SimBa-SAl is more accurate than previously developed pairwise alignment algorithms, when analyzing simulated as well as empirical data sets. Finally, we study the goodness-of-fit of the long-indel and TKF91 models. We show that although the long-indel model fits the data sets better than TKF91, there is still room for improvement concerning the realistic modeling of evolutionary sequence dynamics.

The classic methodology of inferring a phylogenetic tree from sequence data is composed of two steps. First, a multiple sequence alignment (MSA) is computed. Then, a tree is reconstructed assuming the MSA is correct. Yet, inferred MSAs were shown to be inaccurate and alignment errors reduce tree inference accuracy. It was previously proposed that filtering unreliable alignment regions can increase the accuracy of tree inference. However, it was also demonstrated that the benefit of this filtering is often obscured by the resulting loss of phylogenetic signal. In this work we explore an approach, in which instead of relying on a single MSA, we generate a large set of alternative MSAs and concatenate them into a single SuperMSA. By doing so, we account for phylogenetic signals contained in columns that are not present in the single MSA computed by alignment algorithms. Using simulations, we demonstrate that this approach results, on average, in more accurate trees compared to 1) using an unfiltered MSA and 2) using a single MSA with weights assigned to columns according to their reliability. Next, we explore in which regions of the MSA space our approach is expected to be beneficial. Finally, we provide a simple criterion for deciding whether or not the extra effort of computing a SuperMSA and inferring a tree from it is beneficial. Based on these assessments, we expect our methodology to be useful for many cases in which diverged sequences are analyzed. The option to generate such a SuperMSA is available at http://guidance.tau.ac.il.

Peptide-expressing phage display libraries are widely used for the interrogation of antibodies. Affinity selected peptides are then analyzed to discover epitope mimetics, or are subjected to computational algorithms for epitope prediction. A critical assumption for these applications is the random representation of amino acids in the initial naïve peptide library. In a previous study, we implemented next generation sequencing to evaluate a naïve library and discovered severe deviations from randomness in UAG codon over-representation as well as in high G phosphoramidite abundance causing amino acid distribution biases. In this study, we demonstrate that the UAG over-representation can be attributed to the burden imposed on the phage upon the assembly of the recombinant Protein 8 subunits. This was corrected by constructing the libraries using supE44-containing bacteria which suppress the UAG driven abortive termination. We also demonstrate that the overabundance of G stems from variant synthesis-efficiency and can be corrected using compensating oligonucleotide-mixtures calibrated by mass spectroscopy. Construction of libraries implementing these correctives results in markedly improved libraries that display random distribution of amino acids, thus ensuring that enriched peptides obtained in biopanning represent a genuine selection event, a fundamental assumption for phage display applications.