An excess of nonsynonymous over synonymous substitution at individual amino acid sites is an important indicator that positive selection has affected the evolution of a protein between the extant ...sequences under study and their most recent common ancestor. Several methods exist to detect the presence, and sometimes location, of positively selected sites in alignments of protein-coding sequences. This article describes the "sitewise likelihood-ratio" (SLR) method for detecting nonneutral evolution, a statistical test that can identify sites that are unusually conserved as well as those that are unusually variable. We show that the SLR method can be more powerful than currently published methods for detecting the location of positive selection, especially in difficult cases where the strength of selection is low. The increase in power is achieved while relaxing assumptions about how the strength of selection varies over sites and without elevated rates of false-positive results that have been reported with some other methods. We also show that the SLR method performs well even under circumstances where the results from some previous methods can be misleading.
The Monte Carlo simulation of sequence evolution is routinely used to assess the performance of phylogenetic inference methods and sequence alignment algorithms. Progress in the field of molecular ...evolution fuels the need for more realistic and hence more complex simulations, adapted to particular situations, yet current software makes unreasonable assumptions such as homogeneous substitution dynamics or a uniform distribution of indels across the simulated sequences. This calls for an extensible simulation framework written in a high-level functional language, offering new functionality and making it easy to incorporate further complexity.
PhyloSim is an extensible framework for the Monte Carlo simulation of sequence evolution, written in R, using the Gillespie algorithm to integrate the actions of many concurrent processes such as substitutions, insertions and deletions. Uniquely among sequence simulation tools, PhyloSim can simulate arbitrarily complex patterns of rate variation and multiple indel processes, and allows for the incorporation of selective constraints on indel events. User-defined complex patterns of mutation and selection can be easily integrated into simulations, allowing PhyloSim to be adapted to specific needs.
Close integration with R and the wide range of features implemented offer unmatched flexibility, making it possible to simulate sequence evolution under a wide range of realistic settings. We believe that PhyloSim will be useful to future studies involving simulated alignments.
The Exact Call Chemistry for the SOLiD Next-Generation Sequencing platform augments the two-base-encoding chemistry with an additional round of ligation, using an alternative set of probes, that ...allows some mistakes made when reading the first set of probes to be corrected. Additionally, the Exact Call Chemistry allows reads produced by the platform to be decoded directly into nucleotide sequence rather than its two-base 'color' encoding.
We apply the theory of linear codes to analyse the new chemistry, showing the types of sequencing mistakes it can correct and identifying those where the presence of an error can only be detected. For isolated mistakes that cannot be unambiguously corrected, we show that the type of substitution can be determined, and its location can be narrowed down to two or three positions, leading to a significant reduction in the the number of plausible alternative reads.
The Exact Call Chemistry increases the accuracy of the SOLiD platform, enabling many potential miscalls to be prevented. However, single miscalls in the color sequence can produce complex but localised patterns of error in the decoded nucleotide sequence. Analysis of similar codes shows that some exist that, if implemented in alternative chemistries, should have superior performance.
The accumulation of genome-scale molecular data sets for nonmodel taxa brings us ever closer to resolving the tree of life of all living organisms. However, despite the depth of data available, a ...number of studies that each used thousands of genes have reported conflicting results. The focus of phylogenomic projects must thus shift to more careful experimental design. Even though we still have a limited understanding of what are the best predictors of the phylogenetic informativeness of a gene, there is wide agreement that one key factor is its evolutionary rate; but there is no consensus as to whether the rates derived as optimal in various analytical, empirical, and simulation approaches have any general applicability. We here use simulations to infer optimal rates in a set of realistic phylogenetic scenarios with varying tree sizes, numbers of terminals, and tree shapes. Furthermore, we study the relationship between the optimal rate and rate variation among sites and among lineages. Finally, we examine how well the predictions made by a range of experimental design methods correlate with the observed performance in our simulations. We find that the optimal level of divergence is surprisingly robust to differences in taxon sampling and even to among-site and among-lineage rate variation as often encountered in empirical data sets. This finding encourages the use of methods that rely on a single optimal rate to predict a gene's utility. Focusing on correct recovery either of the most basal node in the phylogeny or of the entire topology, the optimal rate is about 0.45 substitutions from root to tip in average Yule trees and about 0.2 in difficult trees with short basal and long-apical branches, but all rates leading to divergence levels between about 0.1 and 0.5 perform reasonably well. Testing the performance of six methods that can be used to predict a gene's utility against our simulation results, we find that the probability of resolution, signal-noise analysis, and Fisher information are good predictors of phylogenetic informativeness, but they require specification of at least part of a model tree. Likelihood quartet mapping also shows very good performance but only requires sequence alignments and is thus applicable without making assumptions about the phylogeny. Despite them being the most commonly used methods for experimental design, geometric quartet mapping and the integration of phylogenetic informativeness curves perform rather poorly in our comparison. Instead of derived predictors of phylogenetic informativeness, we suggest that the number of sites in a gene that evolve at near-optimal rates (as inferred here) could be used directly to prioritize genes for phylogenetic inference. In combination with measures of model fit, especially with respect to compositional biases and among-site and among-lineage rate variation, such an approach has the potential to greatly improve marker choice and should be tested on empirical data.
The rise of Next Generation Sequencing (NGS) technologies has transformed de novo genome sequencing into an accessible research tool, but obtaining high quality eukaryotic genome assemblies remains a ...challenge, mostly due to the abundance of repetitive elements. These also make it difficult to study nucleotide polymorphism in repetitive regions, including certain types of structural variations. One solution proposed for resolving such regions is Sequence Assembly aided by Mutagenesis (SAM), which relies on the fact that introducing enough random mutations breaks the repetitive structure, making assembly possible. Sequencing many different mutated copies permits the sequence of the repetitive region to be inferred by consensus methods. However, this approach relies on molecular cloning in order to isolate and amplify individual mutant copies, making it hard to scale-up the approach for use in conjunction with high-throughput sequencing technologies. To address this problem, we propose NG-SAM, a modified version of the SAM protocol that relies on PCR and dilution steps only, coupled to a NGS workflow. NG-SAM therefore has the potential to be scaled-up, e.g. using emerging microfluidics technologies. We built a realistic simulation pipeline to study the feasibility of NG-SAM, and our results suggest that under appropriate experimental conditions the approach might be successfully put into practice. Moreover, our simulations suggest that NG-SAM is capable of reconstructing robustly a wide range of potential target sequences of varying lengths and repetitive structures.
The accuracy of base calls produced by Illumina sequencers is adversely affected by several processes, with laser cross-talk and cluster phasing being prominent. We introduce an explicit statistical ...model of the sequencing process that generalizes current models of phasing and cross-talk and forms the basis of a base calling method which improves on the best existing base callers, especially when comparing the number of error-free reads. The novel algorithms implemented in All Your Base (AYB) are comparable in speed to other competitive base-calling methods, do not require training data and are designed to be robust to gross errors, producing sensible results where other techniques struggle. AYB is available at http://www.ebi.ac.uk/goldman-srv/AYB/.
The empirical frequencies of DNA k-mers in whole genome sequences provide an interesting perspective on genomic complexity, and the availability of large segments of genomic sequence from many ...organisms means that analysis of k-mers with non-trivial lengths is now possible.
We have studied the k-mer spectra of more than 100 species from Archea, Bacteria, and Eukaryota, particularly looking at the modalities of the distributions. As expected, most species have a unimodal k-mer spectrum. However, a few species, including all mammals, have multimodal spectra. These species coincide with the tetrapods. Genomic sequences are clearly very complex, and cannot be fully explained by any simple probabilistic model. Yet we sought such an explanation for the observed modalities, and discovered that low-order Markov models capture this property (and some others) fairly well.
Multimodal spectra are characterized by specific ranges of values of C+G content and of CpG dinucleotide suppression, a range that encompasses all tetrapods analyzed. Other genomes, like that of the protozoa Entamoeba histolytica, which also exhibits CpG suppression, do not have multimodal k-mer spectra. Groupings of functional elements of the human genome also have a clear modality, and exhibit either a unimodal or multimodal behaviour, depending on the two above mentioned values.
Methods to detect the action of selection on proteins can now make strong predictions about its strength and location, but are becoming increasingly technical. The complexity of the methods makes it ...difficult to determine and interpret the significance of any selection detected. With more information being extracted from the data, the quality of the protein alignment and phylogeny used becomes increasingly important in assessing whether or not a prediction is merely a statistical artifact. Both data quality issues and statistical assessment of the results are considered.
Accurate phylogenetic inference is a topic of intensive research and debate and has been studied in response to many different factors: for example, differences in the method of reconstruction, the ...shape of the underlying tree, the substitution model, and varying quantities and types of data. Investigating whether the conditions used might lead to inaccurate inference has been attempted through elaborate data exploration but less attention has been given to creating a unified methodology to enable experimental designs in phylogenetic analysis to be improved and so avoid suboptimal conditions. Experimental design has been part of the field of statistics since the seminal work of Fisher in the early 20th century and a large body of literature exists on how to design optimum experiments. Here we investigate the use of the Fisher information matrix to decide between candidate positions for adding a taxon to a fixed topology, and introduce a parameter transformation that permits comparison of these different designs. This extension to Goldman (1998. Proc. R. Soc. Lond. B. 265: 1779–1786) thus allows investigation of “where to add taxa” in a phylogeny. We compare three different measures of the total information for selecting the position to add a taxon to a tree. Our methods are illustrated by investigating the behavior of the three criteria when adding a branch to model trees, and by applying the different criteria to two biological examples: a simplified taxon-sampling problem in the balsaminoid Ericales and the phylogeny of seed plants.
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