Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments

Alignment quality may have as much impact on phylogenetic reconstruction as the phylogenetic methods used. Not only the alignment algorithm, but also the method used to deal with the most problematic alignment regions, may have a critical effect on the final tree. Although some authors remove such problematic regions, either manually or using automatic methods, in order to improve phylogenetic performance, others prefer to keep such regions to avoid losing any information. Our aim in the present work was to examine whether phylogenetic reconstruction improves after alignment cleaning or not. Using simulated protein alignments with gaps, we tested the relative performance in diverse phylogenetic analyses of the whole alignments versus the alignments with problematic regions removed with our previously developed Gblocks program. We also tested the performance of more or less stringent conditions in the selection of blocks. Alignments constructed with different alignment methods (ClustalW, Mafft, and Probcons) were used to estimate phylogenetic trees by maximum likelihood, neighbor joining, and parsimony. We show that, in most alignment conditions, and for alignments that are not too short, removal of blocks leads to better trees. That is, despite losing some information, there is an increase in the actual phylogenetic signal. Overall, the best trees are obtained by maximum-likelihood reconstruction of alignments cleaned by Gblocks. In general, a relaxed selection of blocks is better for short alignment, whereas a stringent selection is more adequate for longer ones. Finally, we show that cleaned alignments produce better topologies although, paradoxically, with lower bootstrap. This indicates that divergent and problematic alignment regions may lead, when present, to apparently better supported although, in fact, more biased topologies.     

ClipKIT: A multiple sequence alignment trimming software for accurate phylogenomic inference

Highly divergent sites in multiple sequence alignments (MSAs), which can stem from erroneous inference of homology and saturation of substitutions, are thought to negatively impact phylogenetic inference. Thus, several different trimming strategies have been developed for identifying and removing these sites prior to phylogenetic inference. However, a recent study reported that doing so can worsen inference, underscoring the need for alternative alignment trimming strategies. Here, we introduce ClipKIT, an alignment trimming software that, rather than identifying and removing putatively phylogenetically uninformative sites, instead aims to identify and retain parsimony-informative sites, which are known to be phylogenetically informative. To test the efficacy of ClipKIT, we examined the accuracy and support of phylogenies inferred from 14 different alignment trimming strategies, including those implemented in ClipKIT, across nearly 140,000 alignments from a broad sampling of evolutionary histories. Phylogenies inferred from ClipKIT-trimmed alignments are accurate, robust, and time saving. Furthermore, ClipKIT consistently outperformed other trimming methods across diverse datasets, suggesting that strategies based on identifying and retaining parsimony-informative sites provide a robust framework for alignment trimming.

Highly divergent sites in multiple sequence alignments are thought to negatively impact phylogenetic inference; trimming methods aim to remove these sites, but recent analysis suggests that doing so can worsen inference. This study introduces ClipKIT, a trimming method that instead aims to retain parsimony-informative sites; phylogenetic inference using ClipKIT-trimmed alignments is accurate, robust and time-saving.     

Phylogenetic inference from conserved sites alignments.

Molecular sequences provide a rich source of data for inferring the phylogenetic relationships among species. However, recent work indicates that even an accurate multiple alignment of a large sequence set may yield an incorrect phylogeny and that the quality of the phylogenetic tree improves when the input consists only of the highly conserved, motif regions of the alignment. This work introduces two methods of producing multiple alignments that include only the conserved regions of the initial alignment. The first method retains conserved motifs, whereas the second retains individual conserved sites in the initial alignment. Using parsimony analysis on a mitochondrial data set containing 19 species among which the phylogenetic relationships are widely accepted, both conserved alignment methods produce better phylogenetic trees than the complete alignment. Unlike any of the 19 inference methods used before to analyze this data, both methods produce trees that are completely consistent with the known phylogeny. The motif-based method employs far fewer alignment sites for comparable error rates. For a larger data set containing mitochondrial sequences from 39 species, the site-based method produces a phylogenetic tree that is largely consistent with known phylogenetic relationships and suggests several novel placements. J. Exp. Zool. ( Mol. Dev. Evol.) 285:128-139, 1999.     

Multiple protein sequence alignment

Multiple sequence alignments are essential in computational analysis of protein sequences and structures, with applications in structure modeling, functional site prediction, phylogenetic analysis and sequence database searching. Constructing accurate multiple alignments for divergent protein sequences remains a difficult computational task, and alignment speed becomes an issue for large sequence datasets. Here, I review methodologies and recent advances in the multiple protein sequence alignment field, with emphasis on the use of additional sequence and structural information to improve alignment quality.     

The ancestor of the Bacteria domain was a hyperthermophile

Brochier and Philippe have recently re-analysed the phylogeny of ribosomal RNA using only multiple alignment positions with no phylogenetic noise. They conclude that the first branch of divergence in the Bacteria domain comprises Planctomycetales and not hyperthermophile bacteria as in classic phylogeny. In the present paper I examine the robustness of their conclusions. (1) A site-by-site reading of the RNA alignments of Brochier and Philippe seems to suggest that the number of nucleotide positions used in their analysis is not sufficiently high and their phylogenetic analysis is consequently not robust. Furthermore, (2) a different method for selecting positions with no phylogenetic noise from the rRNA alignment relocates the Aquificales and the Thermotogales as the first lines of divergence in the Bacteria domain, and sets Planctomycetales as the third branch of divergence in the phylogenetic tree built from these selected positions. These findings consolidate the hypothesis that the ancestor of the Bacteria domain was a hyperthermophile and, more generally, that the last universal common ancestor might also have been one.     

No so HoT – heads or tails is not able to reliably compare multiple sequence alignments

Most phylogenetic‐tree building applications use multiple sequence alignments as a starting point. A recent meta‐level methodology, called Heads or Tails, aims to reveal the quality of multiple sequence alignments by comparing alignments taken in the forward direction with the alignments of the same sequences when the sequences are reversed. Through an examination of a special case for multiple sequence alignment – pair‐wise alignments, where an optimal algorithm exists – and the use of a modi?ed global‐alignment application, it is shown that the forward and reverse alignments, even when they are the same, do not capture all the possible variations in the alignments and when the forward and reverse alignments differ there may be other alignments that remain unaccounted for. The implication is that comparing just the forward and (biologically irrelevant) reverse alignments is not sufficient to capture the variability in multiple sequence alignments, and the Heads or Tails methodology is therefore not suitable as a method for investigating multiple sequence alignment accuracy. Part of the reason is the inability of individual multiple sequence alignment applications to adequately sample the space of possible alignments. A further implication is that the Hall [Hall, B.G., 2008. Mol. Biol. Evol. 25, 1576–1580] methodology may create optimal synthetic multiple sequence alignments that extant aligners will be unable to completely recover ab initio due to alternative alignments being possible at particular sites. In general, it is shown that more divergent sequences will give rise to an increased number of alternative alignments, so sequence sets with a higher degree of similarity are preferable to sets with lower similarity as the starting point for phylogenetic tree building. © The Willi Hennig Society 2009.     

Predicting reliable regions in protein sequence alignments

MOTIVATION: Protein sequence alignments have a myriad of applications in bioinformatics, including secondary and tertiary structure prediction, homology modeling, and phylogeny. Unfortunately, all alignment methods make mistakes, and mistakes in alignments often yield mistakes in their application. Thus, a method to identify and remove suspect alignment positions could benefit many areas in protein sequence analysis. RESULTS: We tested four predictors of alignment position reliability, including near-optimal alignment information, column score, and secondary structural information. We validated each predictor against a large library of alignments, removing positions predicted as unreliable. Near-optimal alignment information was the best predictor, removing 70% of the substantially-misaligned positions and 58% of the over-aligned positions, while retaining 86% of those aligned accurately.     

The performance of several multiple-sequence alignment programs in relation to secondary-structure features for an rRNA sequence

The performances of five global multiple-sequence alignment programs (CLUSTAL W, Divide and Conquer, Malign, PileUp, and TreeAlign) were evaluated using part of the animal mitochondrial small subunit (12S) rRNA molecule. Conserved sequence motifs derived from an alignment based on secondary structural information were used to score how well each program aligned a data set of five vertebrate and five invertebrate taxa over a range of parameter values. All of the programs could align the motifs with reasonable accuracy for at least one set of parameter conditions, although if the whole sequence was considered, similarity to the structural alignment was only 25%-34%. Use of small gap costs generally gave more accurate results, although Malign and TreeAlign generated longer alignments when gap costs were low. The programs differed in the consistency of the alignments when gap cost was varied; CLUSTAL W, Divide and Conquer, and TreeAlign were the most accurate and robust, while PileUp performed poorly as gap cost values increased, and the accuracy of Malign fluctuated. Default settings for the programs did not give the best results, and attempting to select similar parameter values in different programs did not always result in more similar alignments. Poor alignment of even well-conserved motifs can occur if these are near sites with insertions or deletions. Since there is no a priori way to determine gap costs and because such costs can vary over the gene, alignment of rRNA sequences, particularly the less well conserved regions, should be treated carefully and aided by secondary structure and conserved motifs. Some motifs are single bases and so are often invisible to alignment programs. Our tests involved the most conserved regions of the 12S rRNA gene, and alignment of less well conserved regions will be more problematical. None of the alignments we examined produced a fully resolved phylogeny for the data set, indicating that this portion of 12S rRNA is insufficient for resolution of distant evolutionary relationships.     

MAVID multiple alignment server

MAVID is a multiple alignment program suitable for many large genomic regions. The MAVID web server allows biomedical researchers to quickly obtain multiple alignments for genomic sequences and to subsequently analyse the alignments for conserved regions. MAVID has been successfully used for the alignment of closely related species such as primates and also for the alignment of more distant organisms such as human and fugu. The server is fast, capable of aligning hundreds of kilobases in less than a minute. The multiple alignment is used to build a phylogenetic tree for the sequences, which is subsequently used as a basis for identifying conserved regions in the alignment. The server can be accessed at http://baboon.math.berkeley.edu/mavid/.     

Phylogeny of Coreopsideae (Asteraceae) using ITS sequences suggests lability in reproductive characters

Relationships among the 21 genera within the tribe Coreopsideae (Asteraceae) remain poorly resolved despite phylogenetic studies using morphological and anatomical traits. Recent molecular phylogenies have also indicated that some Coreopsideae genera are not monophyletic. We used internal transcribed spacer (ITS) sequences from representatives of 19 genera, as well as all major lineages in those genera that are not monophyletic, to examine phylogenetic relationships within this group. To examine the affects of alignment and method of analysis on our conclusions, we obtained alignments using five different parameters and analyzed all five alignments with distance, parsimony, and Bayesian methods. The method of analysis had a larger impact on relationships than did alignments, although different analytical methods gave very similar results. Although not all relationships could be resolved, a number of well-supported lineages were found, some in conflict with earlier hypotheses. We did not find monophyly in Bidens, Coreopsis, and Coreocarpus, though other genera were monophyletic for the taxa we included. Morphological and anatomical traits which have been used previously to resolve phylogenetic relationships in this group were mapped onto the well-supported nodes of the ITS phylogeny. This analysis indicated that floral and fruit characters, which have been used extensively in phylogenetic studies in the Coreopsideae, show a higher degree of evolutionary lability in this group than the more highly conserved vegetative and photosynthetic traits.     

Alignments of mitochondrial genome arrangements: applications to metazoan phylogeny

Mitochondrial genomes provide a valuable dataset for phylogenetic studies, in particular of metazoan phylogeny because of the extensive taxon sample that is available. Beyond the traditional sequence-based analysis it is possible to extract phylogenetic information from the gene order. Here we present a novel approach utilizing these data based on cyclic list alignments of the gene orders. A progressive alignment approach is used to combine pairwise list alignments into a multiple alignment of gene orders. Parsimony methods are used to reconstruct phylogenetic trees, ancestral gene orders, and consensus patterns in a straightforward approach. We apply this method to study the phylogeny of protostomes based exclusively on mitochondrial genome arrangements. We, furthermore, demonstrate that our approach is also applicable to the much larger genomes of chloroplasts.     

Incorporation of gap characters and lineage-specific regions into phylogenetic analyses of gene families from divergent clades: an example from the kinesin superfamily across eukaryotes

The kinesin superfamily across eukaryotes was used to examine how incorporation of gap characters scored from conserved regions shared by all members of a gene family and incorporation of amino acid and gap characters scored from lineage‐specific regions affect gene‐tree inference of the gene family as a whole. We addressed these two questions in the context of two different densities of sequence sampling, four alignment programs, and two methods of tree construction. Taken together, our findings suggest the following. First, gap characters should be incorporated into gene‐tree inference, even for divergent sequences. Second, gene regions that are not conserved among all or most sequences sampled should not be automatically discarded without evaluation of potential phylogenetic signal that may be contained in gap and/or sequence characters. Third, among the four alignment programs evaluated using their default alignment parameters, Clustal may be expected to output alignments that result in the greatest gene‐tree resolution and support. Yet, this high resolution and support should be regarded as optimistic, rather than conservative, estimates. Fourth, this same conclusion regarding resolution and support holds for Bayesian gene‐tree analyses relative to parsimony‐jackknife gene‐tree analyses. We suggest that a more conservative approach, such as aligning the sequences using DIALIGN‐T or MAFFT, analyzing the appropriate characters using parsimony, and assessing branch support using the jackknife, is more appropriate for inferring gene trees of divergent gene families. © The Willi Hennig Society 2007.     

AL2CO: calculation of positional conservation in a protein sequence alignment

MOTIVATION: Amino acid sequence alignments are widely used in the analysis of protein structure, function and evolutionary relationships. Proteins within a superfamily usually share the same fold and possess related functions. These structural and functional constraints are reflected in the alignment conservation patterns. Positions of functional and/or structural importance tend to be more conserved. Conserved positions are usually clustered in distinct motifs surrounded by sequence segments of low conservation. Poorly conserved regions might also arise from the imperfections in multiple alignment algorithms and thus indicate possible alignment errors. Quantification of conservation by attributing a conservation index to each aligned position makes motif detection more convenient. Mapping these conservation indices onto a protein spatial structure helps to visualize spatial conservation features of the molecule and to predict functionally and/or structurally important sites. Analysis of conservation indices could be a useful tool in detection of potentially misaligned regions and will aid in improvement of multiple alignments. RESULTS: We developed a program to calculate a conservation index at each position in a multiple sequence alignment using several methods. Namely, amino acid frequencies at each position are estimated and the conservation index is calculated from these frequencies. We utilize both unweighted frequencies and frequencies weighted using two different strategies. Three conceptually different approaches (entropy-based, variance-based and matrix score-based) are implemented in the algorithm to define the conservation index. Calculating conservation indices for 35522 positions in 284 alignments from SMART database we demonstrate that different methods result in highly correlated (correlation coefficient more than 0.85) conservation indices. Conservation indices show statistically significant correlation between sequentially adjacent positions i and i + j, where j < 13, and averaging of the indices over the window of three positions is optimal for motif detection. Positions with gaps display substantially lower conservation properties. We compare conservation properties of the SMART alignments or FSSP structural alignments to those of the ClustalW alignments. The results suggest that conservation indices should be a valuable tool of alignment quality assessment and might be used as an objective function for refinement of multiple alignments. AVAILABILITY: The C code of the AL2CO program and its pre-compiled versions for several platforms as well as the details of the analysis are freely available at ftp://iole.swmed.edu/pub/al2co/.     

Re-Mind the Gap! Insertion – Deletion Data Reveal Neglected Phylogenetic Potential of the Nuclear Ribosomal Internal Transcribed Spacer (ITS) of Fungi

Rapidly evolving, indel-rich phylogenetic markers play a pivotal role in our understanding of the relationships at multiple levels of the tree of life. There is extensive evidence that indels provide conserved phylogenetic signal, however, the range of phylogenetic depths for which gaps retain tree signal has not been investigated in detail. Here we address this question using the fungal internal transcribed spacer (ITS), which is central in many phylogenetic studies, molecular ecology, detection and identification of pathogenic and non-pathogenic species. ITS is repeatedly criticized for indel-induced alignment problems and the lack of phylogenetic resolution above species level, although these have not been critically investigated. In this study, we examined whether the inclusion of gap characters in the analyses shifts the phylogenetic utility of ITS alignments towards earlier divergences. By re-analyzing 115 published fungal ITS alignments, we found that indels are slightly more conserved than nucleotide substitutions, and when included in phylogenetic analyses, improved the resolution and branch support of phylogenies across an array of taxonomic ranges and extended the resolving power of ITS towards earlier nodes of phylogenetic trees. Our results reconcile previous contradicting evidence for the effects of data exclusion: in the case of more sophisticated indel placement, the exclusion of indel-rich regions from the analyses results in a loss of tree resolution, whereas in the case of simpler alignment methods, the exclusion of gapped sites improves it. Although the empirical datasets do not provide to measure alignment accuracy objectively, our results for the ITS region are consistent with previous simulations studies alignment algorithms. We suggest that sophisticated alignment algorithms and the inclusion of indels make the ITS region and potentially other rapidly evolving indel-rich loci valuable sources of phylogenetic information, which can be exploited at multiple taxonomic levels.     

Accounting for alignment uncertainty in phylogenomics

Uncertainty in multiple sequence alignments has a large impact on phylogenetic analyses. Little has been done to evaluate the quality of individual positions in protein sequence alignments, which directly impact the accuracy of phylogenetic trees. Here we describe ZORRO, a probabilistic masking program that accounts for alignment uncertainty by assigning confidence scores to each alignment position. Using the BALIBASE database and in simulation studies, we demonstrate that masking by ZORRO significantly reduces the alignment uncertainty and improves the tree accuracy.     

Multiple protein structure alignment.

A method was developed to compare protein structures and to combine them into a multiple structure consensus. Previous methods of multiple structure comparison have only concatenated pairwise alignments or produced a consensus structure by averaging coordinate sets. The current method is a fusion of the fast structure comparison program SSAP and the multiple sequence alignment program MULTAL. As in MULTAL, structures are progressively combined, producing intermediate consensus structures that are compared directly to each other and all remaining single structures. This leads to a hierarchic "condensation," continually evaluated in the light of the emerging conserved core regions. Following the SSAP approach, all interatomic vectors were retained with well-conserved regions distinguished by coherent vector bundles (the structural equivalent of a conserved sequence position). Each bundle of vectors is summarized by a resultant, whereas vector coherence is captured in an error term, which is the only distinction between conserved and variable positions. Resultant vectors are used directly in the comparison, which is weighted by their error values, giving greater importance to the matching of conserved positions. The resultant vectors and their errors can also be used directly in molecular modeling. Applications of the method were assessed by the quality of the resulting sequence alignments, phylogenetic tree construction, and databank scanning with the consensus. Visual assessment of the structural superpositions and consensus structure for various well-characterized families confirmed that the consensus had identified a reasonable core.     

PHYRN: a robust method for phylogenetic analysis of highly divergent sequences

Both multiple sequence alignment and phylogenetic analysis are problematic in the "twilight zone" of sequence similarity (≤ 25% amino acid identity). Herein we explore the accuracy of phylogenetic inference at extreme sequence divergence using a variety of simulated data sets. We evaluate four leading multiple sequence alignment (MSA) methods (MAFFT, T-COFFEE, CLUSTAL, and MUSCLE) and six commonly used programs of tree estimation (Distance-based: Neighbor-Joining; Character-based: PhyML, RAxML, GARLI, Maximum Parsimony, and Bayesian) against a novel MSA-independent method (PHYRN) described here. Strikingly, at "midnight zone" genetic distances (~7% pairwise identity and 4.0 gaps per position), PHYRN returns high-resolution phylogenies that outperform traditional approaches. We reason this is due to PHRYN's capability to amplify informative positions, even at the most extreme levels of sequence divergence. We also assess the applicability of the PHYRN algorithm for inferring deep evolutionary relationships in the divergent DANGER protein superfamily, for which PHYRN infers a more robust tree compared to MSA-based approaches. Taken together, these results demonstrate that PHYRN represents a powerful mechanism for mapping uncharted frontiers in highly divergent protein sequence data sets.     

Ribosomal protein-sequence block structure suggests complex prokaryotic evolution with implications for the origin of eukaryotes

Amino acid sequence alignments of orthologous ribosomal proteins found in Bacteria, Archaea, and Eukaryota display, relative to one another, an unusual segment or block structure, with major evolutionary implications. Within each of the prokaryotic phylodomains the sequences exhibit substantial similarity, but cross-domain alignments break up into (a) universal blocks (conserved in both phylodomains), (b) bacterial blocks (unalignable with any archaeal counterparts), and (c) archaeal blocks (unalignable with any bacterial counterparts). Sequences of those eukaryotic cytoplasmic riboproteins that have orthologs in both Bacteria and Archaea, exclusively match the archaeal block structure. The distinct blocks do not correlate consistently with any identifiable functional or structural feature including RNA and protein contacts. This phylodomain-specific block pattern also exists in a number of other proteins associated with protein synthesis, but not among enzymes of intermediary metabolism. While the universal blocks imply that modern Bacteria and Archaea (as defined by their translational machinery) clearly have had a common ancestor, the phylodomain-specific blocks imply that these two groups derive from single, phylodomain-specific types that came into existence at some point long after that common ancestor. The simplest explanation for this pattern would be a major evolutionary bottleneck, or other scenario that drastically limited the progenitors of modern prokaryotic diversity at a time considerably after the evolution of a fully functional translation apparatus. The vast range of habitats and metabolisms that prokaryotes occupy today would thus reflect divergent evolution after such a restricting event. Interestingly, phylogenetic analysis places the origin of eukaryotes at about the same time and shows a closer relationship of the eukaryotic ribosome-associated proteins to crenarchaeal rather than euryarchaeal counterparts.     

New features of the Blocks Database servers.

Blocks are ungapped multiple sequence alignments representing conserved protein regions, and the Blocks Database consists of blocks from documented protein families. World Wide Web (http://www. blocks.fhcrc.org) and Email (blocks@blocks.fhcrc.org) servers provide tools for homology searching and for analyzing protein family relationships. New enhancements include a multiple alignment processor that extends the use of these tools to imported multiple alignments of families not present in the database and a PCR primer designer that implements a new strategy for gene isolation.     

Phylogeny-independent detection of functional residues

MOTIVATION: Current projects for the massive characterization of proteomes are generating protein sequences and structures with unknown function. The difficulty of experimentally determining functionally important sites calls for the development of computational methods. The first techniques, based on the search for fully conserved positions in multiple sequence alignments (MSAs), were followed by methods for locating family-dependent conserved positions. These rely on the functional classification implicit in the alignment for locating these positions related with functional specificity. The next obvious step, still scarcely explored, is to detect these positions using a functional classification different from the one implicit in the sequence relationships between the proteins. Here, we present two new methods for locating functional positions which can incorporate an arbitrary external functional classification which may or may not coincide with the one implicit in the MSA. The Xdet method is able to use a functional classification with an associated hierarchy or similarity between functions to locate positions related to that classification. The MCdet method uses multivariate statistical analysis to locate positions responsible for each one of the functions within a multifunctional family. RESULTS: We applied the methods to different cases, illustrating scenarios where there is a disagreement between the functional and the phylogenetic relationships, and demonstrated their usefulness for the phylogeny-independent prediction of functional positions.