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1.  PicXAA: greedy probabilistic construction of maximum expected accuracy alignment of multiple sequences 
Nucleic Acids Research  2010;38(15):4917-4928.
Accurate tools for multiple sequence alignment (MSA) are essential for comparative studies of the function and structure of biological sequences. However, it is very challenging to develop a computationally efficient algorithm that can consistently predict accurate alignments for various types of sequence sets. In this article, we introduce PicXAA (Probabilistic Maximum Accuracy Alignment), a probabilistic non-progressive alignment algorithm that aims to find protein alignments with maximum expected accuracy. PicXAA greedily builds up the multiple alignment from sequence regions with high local similarities, thereby yielding an accurate global alignment that effectively grasps the local similarities among sequences. Evaluations on several widely used benchmark sets show that PicXAA constantly yields accurate alignment results on a wide range of reference sets, with especially remarkable improvements over other leading algorithms on sequence sets with local similarities. PicXAA source code is freely available at:∼bjyoon/picxaa/.
PMCID: PMC2926610  PMID: 20413579
2.  PROMALS3D: a tool for multiple protein sequence and structure alignments 
Nucleic Acids Research  2008;36(7):2295-2300.
Although multiple sequence alignments (MSAs) are essential for a wide range of applications from structure modeling to prediction of functional sites, construction of accurate MSAs for distantly related proteins remains a largely unsolved problem. The rapidly increasing database of spatial structures is a valuable source to improve alignment quality. We explore the use of 3D structural information to guide sequence alignments constructed by our MSA program PROMALS. The resulting tool, PROMALS3D, automatically identifies homologs with known 3D structures for the input sequences, derives structural constraints through structure-based alignments and combines them with sequence constraints to construct consistency-based multiple sequence alignments. The output is a consensus alignment that brings together sequence and structural information about input proteins and their homologs. PROMALS3D can also align sequences of multiple input structures, with the output representing a multiple structure-based alignment refined in combination with sequence constraints. The advantage of PROMALS3D is that it gives researchers an easy way to produce high-quality alignments consistent with both sequences and structures of proteins. PROMALS3D outperforms a number of existing methods for constructing multiple sequence or structural alignments using both reference-dependent and reference-independent evaluation methods.
PMCID: PMC2367709  PMID: 18287115
3.  Efficient protein alignment algorithm for protein search 
BMC Bioinformatics  2010;11(Suppl 1):S34.
Proteins show a great variety of 3D conformations, which can be used to infer their evolutionary relationship and to classify them into more general groups; therefore protein structure alignment algorithms are very helpful for protein biologists. However, an accurate alignment algorithm itself may be insufficient for effective discovering of structural relationships among tens of thousands of proteins. Due to the exponentially increasing amount of protein structural data, a fast and accurate structure alignment tool is necessary to access protein classification and protein similarity search; however, the complexity of current alignment algorithms are usually too high to make a fully alignment-based classification and search practical.
We have developed an efficient protein pairwise alignment algorithm and applied it to our protein search tool, which aligns a query protein structure in the pairwise manner with all protein structures in the Protein Data Bank (PDB) to output similar protein structures. The algorithm can align hundreds of pairs of protein structures in one second. Given a protein structure, the tool efficiently discovers similar structures from tens of thousands of structures stored in the PDB always in 2 minutes in a single machine and 20 seconds in our cluster of 6 machines. The algorithm has been fully implemented and is accessible online at our webserver, which is supported by a cluster of computers.
Our algorithm can work out hundreds of pairs of protein alignments in one second. Therefore, it is very suitable for protein search. Our experimental results show that it is more accurate than other well known protein search systems in finding proteins which are structurally similar at SCOP family and superfamily levels, and its speed is also competitive with those systems. In terms of the pairwise alignment performance, it is as good as some well known alignment algorithms.
PMCID: PMC3009506  PMID: 20122207
4.  Rapid detection, classification and accurate alignment of up to a million or more related protein sequences 
Bioinformatics  2009;25(15):1869-1875.
Motivation: The patterns of sequence similarity and divergence present within functionally diverse, evolutionarily related proteins contain implicit information about corresponding biochemical similarities and differences. A first step toward accessing such information is to statistically analyze these patterns, which, in turn, requires that one first identify and accurately align a very large set of protein sequences. Ideally, the set should include many distantly related, functionally divergent subgroups. Because it is extremely difficult, if not impossible for fully automated methods to align such sequences correctly, researchers often resort to manual curation based on detailed structural and biochemical information. However, multiply-aligning vast numbers of sequences in this way is clearly impractical.
Results: This problem is addressed using Multiply-Aligned Profiles for Global Alignment of Protein Sequences (MAPGAPS). The MAPGAPS program uses a set of multiply-aligned profiles both as a query to detect and classify related sequences and as a template to multiply-align the sequences. It relies on Karlin–Altschul statistics for sensitivity and on PSI-BLAST (and other) heuristics for speed. Using as input a carefully curated multiple-profile alignment for P-loop GTPases, MAPGAPS correctly aligned weakly conserved sequence motifs within 33 distantly related GTPases of known structure. By comparison, the sequence- and structurally based alignment methods hmmalign and PROMALS3D misaligned at least 11 and 23 of these regions, respectively. When applied to a dataset of 65 million protein sequences, MAPGAPS identified, classified and aligned (with comparable accuracy) nearly half a million putative P-loop GTPase sequences.
Availability: A C++ implementation of MAPGAPS is available at
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2732367  PMID: 19505947
5.  Evolutionary inaccuracy of pairwise structural alignments 
Bioinformatics  2012;28(9):1209-1215.
Motivation: Structural alignment methods are widely used to generate gold standard alignments for improving multiple sequence alignments and transferring functional annotations, as well as for assigning structural distances between proteins. However, the correctness of the alignments generated by these methods is difficult to assess objectively since little is known about the exact evolutionary history of most proteins. Since homology is an equivalence relation, an upper bound on alignment quality can be found by assessing the consistency of alignments. Measuring the consistency of current methods of structure alignment and determining the causes of inconsistencies can, therefore, provide information on the quality of current methods and suggest possibilities for further improvement.
Results: We analyze the self-consistency of seven widely-used structural alignment methods (SAP, TM-align, Fr-TM-align, MAMMOTH, DALI, CE and FATCAT) on a diverse, non-redundant set of 1863 domains from the SCOP database and demonstrate that even for relatively similar proteins the degree of inconsistency of the alignments on a residue level is high (30%). We further show that levels of consistency vary substantially between methods, with two methods (SAP and Fr-TM-align) producing more consistent alignments than the rest. Inconsistency is found to be higher near gaps and for proteins of low structural complexity, as well as for helices. The ability of the methods to identify good structural alignments is also assessed using geometric measures, for which FATCAT (flexible mode) is found to be the best performer despite being highly inconsistent. We conclude that there is substantial scope for improving the consistency of structural alignment methods.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3338010  PMID: 22399676
6.  AlignHUSH: Alignment of HMMs using structure and hydrophobicity information 
BMC Bioinformatics  2011;12:275.
Sensitive remote homology detection and accurate alignments especially in the midnight zone of sequence similarity are needed for better function annotation and structural modeling of proteins. An algorithm, AlignHUSH for HMM-HMM alignment has been developed which is capable of recognizing distantly related domain families The method uses structural information, in the form of predicted secondary structure probabilities, and hydrophobicity of amino acids to align HMMs of two sets of aligned sequences. The effect of using adjoining column(s) information has also been investigated and is found to increase the sensitivity of HMM-HMM alignments and remote homology detection.
We have assessed the performance of AlignHUSH using known evolutionary relationships available in SCOP. AlignHUSH performs better than the best HMM-HMM alignment methods and is observed to be even more sensitive at higher error rates. Accuracy of the alignments obtained using AlignHUSH has been assessed using the structure-based alignments available in BaliBASE. The alignment length and the alignment quality are found to be appropriate for homology modeling and function annotation. The alignment accuracy is found to be comparable to existing methods for profile-profile alignments.
A new method to align HMMs has been developed and is shown to have better sensitivity at error rates of 10% and above when compared to other available programs. The proposed method could effectively aid obtaining clues to functions of proteins of yet unknown function.
A web-server incorporating the AlignHUSH method is available at
PMCID: PMC3228556  PMID: 21729312
7.  Protein structure alignment beyond spatial proximity 
Scientific Reports  2013;3:1448.
Protein structure alignment is a fundamental problem in computational structure biology. Many programs have been developed for automatic protein structure alignment, but most of them align two protein structures purely based upon geometric similarity without considering evolutionary and functional relationship. As such, these programs may generate structure alignments which are not very biologically meaningful from the evolutionary perspective. This paper presents a novel method DeepAlign for automatic pairwise protein structure alignment. DeepAlign aligns two protein structures using not only spatial proximity of equivalent residues (after rigid-body superposition), but also evolutionary relationship and hydrogen-bonding similarity. Experimental results show that DeepAlign can generate structure alignments much more consistent with manually-curated alignments than other automatic tools especially when proteins under consideration are remote homologs. These results imply that in addition to geometric similarity, evolutionary information and hydrogen-bonding similarity are essential to aligning two protein structures.
PMCID: PMC3596798  PMID: 23486213
8.  High quality protein sequence alignment by combining structural profile prediction and profile alignment using SABERTOOTH 
BMC Bioinformatics  2010;11:251.
Protein alignments are an essential tool for many bioinformatics analyses. While sequence alignments are accurate for proteins of high sequence similarity, they become unreliable as they approach the so-called 'twilight zone' where sequence similarity gets indistinguishable from random. For such distant pairs, structure alignment is of much better quality. Nevertheless, sequence alignment is the only choice in the majority of cases where structural data is not available. This situation demands development of methods that extend the applicability of accurate sequence alignment to distantly related proteins.
We develop a sequence alignment method that combines the prediction of a structural profile based on the protein's sequence with the alignment of that profile using our recently published alignment tool SABERTOOTH. In particular, we predict the contact vector of protein structures using an artificial neural network based on position-specific scoring matrices generated by PSI-BLAST and align these predicted contact vectors. The resulting sequence alignments are assessed using two different tests: First, we assess the alignment quality by measuring the derived structural similarity for cases in which structures are available. In a second test, we quantify the ability of the significance score of the alignments to recognize structural and evolutionary relationships. As a benchmark we use a representative set of the SCOP (structural classification of proteins) database, with similarities ranging from closely related proteins at SCOP family level, to very distantly related proteins at SCOP fold level. Comparing these results with some prominent sequence alignment tools, we find that SABERTOOTH produces sequence alignments of better quality than those of Clustal W, T-Coffee, MUSCLE, and PSI-BLAST. HHpred, one of the most sophisticated and computationally expensive tools available, outperforms our alignment algorithm at family and superfamily levels, while the use of SABERTOOTH is advantageous for alignments at fold level. Our alignment scheme will profit from future improvements of structural profiles prediction.
We present the automatic sequence alignment tool SABERTOOTH that computes pairwise sequence alignments of very high quality. SABERTOOTH is especially advantageous when applied to alignments of remotely related proteins. The source code is available at, free for academic users upon request.
PMCID: PMC2885375  PMID: 20470364
9.  R-Coffee: a web server for accurately aligning noncoding RNA sequences 
Nucleic Acids Research  2008;36(Web Server issue):W10-W13.
The R-Coffee web server produces highly accurate multiple alignments of noncoding RNA (ncRNA) sequences, taking into account predicted secondary structures. R-Coffee uses a novel algorithm recently incorporated in the T-Coffee package. R-Coffee works along the same lines as T-Coffee: it uses pairwise or multiple sequence alignment (MSA) methods to compute a primary library of input alignments. The program then computes an MSA highly consistent with both the alignments contained in the library and the secondary structures associated with the sequences. The secondary structures are predicted using RNAplfold. The server provides two modes. The slow/accurate mode is restricted to small datasets (less than 5 sequences less than 150 nucleotides) and combines R-Coffee with Consan, a very accurate pairwise RNA alignment method. For larger datasets a fast method can be used (RM-Coffee mode), that uses R-Coffee to combine the output of the three packages which combines the outputs from programs found to perform best on RNA (MUSCLE, MAFFT and ProbConsRNA). Our BRAliBase benchmarks indicate that the R-Coffee/Consan combination is one of the best ncRNA alignment methods for short sequences, while the RM-Coffee gives comparable results on longer sequences. The R-Coffee web server is available at
PMCID: PMC2447777  PMID: 18483080
10.  Protein threading using context-specific alignment potential 
Bioinformatics  2013;29(13):i257-i265.
Motivation: Template-based modeling, including homology modeling and protein threading, is the most reliable method for protein 3D structure prediction. However, alignment errors and template selection are still the main bottleneck for current template-base modeling methods, especially when proteins under consideration are distantly related.
Results: We present a novel context-specific alignment potential for protein threading, including alignment and template selection. Our alignment potential measures the log-odds ratio of one alignment being generated from two related proteins to being generated from two unrelated proteins, by integrating both local and global context-specific information. The local alignment potential quantifies how well one sequence residue can be aligned to one template residue based on context-specific information of the residues. The global alignment potential quantifies how well two sequence residues can be placed into two template positions at a given distance, again based on context-specific information. By accounting for correlation among a variety of protein features and making use of context-specific information, our alignment potential is much more sensitive than the widely used context-independent or profile-based scoring function. Experimental results confirm that our method generates significantly better alignments and threading results than the best profile-based methods on several large benchmarks. Our method works particularly well for distantly related proteins or proteins with sparse sequence profiles because of the effective integration of context-specific, structure and global information.
PMCID: PMC3694651  PMID: 23812991
11.  Improving pairwise sequence alignment accuracy using near-optimal protein sequence alignments 
BMC Bioinformatics  2010;11:146.
While the pairwise alignments produced by sequence similarity searches are a powerful tool for identifying homologous proteins - proteins that share a common ancestor and a similar structure; pairwise sequence alignments often fail to represent accurately the structural alignments inferred from three-dimensional coordinates. Since sequence alignment algorithms produce optimal alignments, the best structural alignments must reflect suboptimal sequence alignment scores. Thus, we have examined a range of suboptimal sequence alignments and a range of scoring parameters to understand better which sequence alignments are likely to be more structurally accurate.
We compared near-optimal protein sequence alignments produced by the Zuker algorithm and a set of probabilistic alignments produced by the probA program with structural alignments produced by four different structure alignment algorithms. There is significant overlap between the solution spaces of structural alignments and both the near-optimal sequence alignments produced by commonly used scoring parameters for sequences that share significant sequence similarity (E-values < 10-5) and the ensemble of probA alignments. We constructed a logistic regression model incorporating three input variables derived from sets of near-optimal alignments: robustness, edge frequency, and maximum bits-per-position. A ROC analysis shows that this model more accurately classifies amino acid pairs (edges in the alignment path graph) according to the likelihood of appearance in structural alignments than the robustness score alone. We investigated various trimming protocols for removing incorrect edges from the optimal sequence alignment; the most effective protocol is to remove matches from the semi-global optimal alignment that are outside the boundaries of the local alignment, although trimming according to the model-generated probabilities achieves a similar level of improvement. The model can also be used to generate novel alignments by using the probabilities in lieu of a scoring matrix. These alignments are typically better than the optimal sequence alignment, and include novel correct structural edges. We find that the probA alignments sample a larger variety of alignments than the Zuker set, which more frequently results in alignments that are closer to the structural alignments, but that using the probA alignments as input to the regression model does not increase performance.
The pool of suboptimal pairwise protein sequence alignments substantially overlaps structure-based alignments for pairs with statistically significant similarity, and a regression model based on information contained in this alignment pool improves the accuracy of pairwise alignments with respect to structure-based alignments.
PMCID: PMC2850363  PMID: 20307279
12.  T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension 
Nucleic Acids Research  2011;39(Web Server issue):W13-W17.
This article introduces a new interface for T-Coffee, a consistency-based multiple sequence alignment program. This interface provides an easy and intuitive access to the most popular functionality of the package. These include the default T-Coffee mode for protein and nucleic acid sequences, the M-Coffee mode that allows combining the output of any other aligners, and template-based modes of T-Coffee that deliver high accuracy alignments while using structural or homology derived templates. These three available template modes are Expresso for the alignment of protein with a known 3D-Structure, R-Coffee to align RNA sequences with conserved secondary structures and PSI-Coffee to accurately align distantly related sequences using homology extension. The new server benefits from recent improvements of the T-Coffee algorithm and can align up to 150 sequences as long as 10 000 residues and is available from both and its main mirror
PMCID: PMC3125728  PMID: 21558174
13.  Application of protein structure alignments to iterated hidden Markov model protocols for structure prediction 
BMC Bioinformatics  2006;7:410.
One of the most powerful methods for the prediction of protein structure from sequence information alone is the iterative construction of profile-type models. Because profiles are built from sequence alignments, the sequences included in the alignment and the method used to align them will be important to the sensitivity of the resulting profile. The inclusion of highly diverse sequences will presumably produce a more powerful profile, but distantly related sequences can be difficult to align accurately using only sequence information. Therefore, it would be expected that the use of protein structure alignments to improve the selection and alignment of diverse sequence homologs might yield improved profiles. However, the actual utility of such an approach has remained unclear.
We explored several iterative protocols for the generation of profile hidden Markov models. These protocols were tailored to allow the inclusion of protein structure alignments in the process, and were used for large-scale creation and benchmarking of structure alignment-enhanced models. We found that models using structure alignments did not provide an overall improvement over sequence-only models for superfamily-level structure predictions. However, the results also revealed that the structure alignment-enhanced models were complimentary to the sequence-only models, particularly at the edge of the "twilight zone". When the two sets of models were combined, they provided improved results over sequence-only models alone. In addition, we found that the beneficial effects of the structure alignment-enhanced models could not be realized if the structure-based alignments were replaced with sequence-based alignments. Our experiments with different iterative protocols for sequence-only models also suggested that simple protocol modifications were unable to yield equivalent improvements to those provided by the structure alignment-enhanced models. Finally, we found that models using structure alignments provided fold-level structure assignments that were superior to those produced by sequence-only models.
When attempting to predict the structure of remote homologs, we advocate a combined approach in which both traditional models and models incorporating structure alignments are used.
PMCID: PMC1622756  PMID: 16970830
14.  PicXAA-Web: a web-based platform for non-progressive maximum expected accuracy alignment of multiple biological sequences 
Nucleic Acids Research  2011;39(Web Server issue):W8-W12.
In this article, we introduce PicXAA-Web, a web-based platform for accurate probabilistic alignment of multiple biological sequences. The core of PicXAA-Web consists of PicXAA, a multiple protein/DNA sequence alignment algorithm, and PicXAA-R, an extension of PicXAA for structural alignment of RNA sequences. Both PicXAA and PicXAA-R are probabilistic non-progressive alignment algorithms that aim to find the optimal alignment of multiple biological sequences by maximizing the expected accuracy. PicXAA and PicXAA-R greedily build up the alignment from sequence regions with high local similarity, thereby yielding an accurate global alignment that effectively captures local similarities among sequences. PicXAA-Web integrates these two algorithms in a user-friendly web platform for accurate alignment and analysis of multiple protein, DNA and RNA sequences. PicXAA-Web can be freely accessed at
PMCID: PMC3125727  PMID: 21515632
15.  Biclustering as a method for RNA local multiple sequence alignment 
Bioinformatics (Oxford, England)  2007;23(24):3289-3296.
Biclustering is a clustering method that simultaneously clusters both the domain and range of a relation. A challenge in multiple sequence alignment (MSA) is that the alignment of sequences is often intended to reveal groups of conserved functional subsequences. Simultaneously, the grouping of the sequences can impact the alignment; precisely the kind of dual situation biclustering is intended to address.
We define a representation of the MSA problem enabling the application of biclustering algorithms. We develop a computer program for local MSA, BlockMSA, that combines biclustering with divide-and-conquer. BlockMSA simultaneously finds groups of similar sequences and locally aligns subsequences within them. Further alignment is accomplished by dividing both the set of sequences and their contents. The net result is both a multiple sequence alignment and a hierarchical clustering of the sequences. BlockMSA was tested on the subsets of the BRAliBase 2.1 benchmark suite that display high variability and on an extension to that suite to larger problem sizes. Also, alignments were evaluated of two large datasets of current biological interest, T box sequences and Group IC1 Introns. The results were compared with alignments computed by ClustalW, MAFFT, MUCLE and PROBCONS alignment programs using Sum of Pairs (SPS) and Consensus Count.
Results for the benchmark suite are sensitive to problem size. On problems of 15 or greater sequences, BlockMSA is consistently the best. On none of the problems in the test suite are there appreciable differences in scores among BlockMSA, MAFFT and PROBCONS. On the T box sequences, BlockMSA does the most faithful job of reproducing known annotations. MAFFT and PROBCONS do not. On the Intron sequences, BlockMSA, MAFFT and MUSCLE are comparable at identifying conserved regions.
PMCID: PMC2228335  PMID: 17921494
16.  An Alignment Confidence Score Capturing Robustness to Guide Tree Uncertainty 
Molecular Biology and Evolution  2010;27(8):1759-1767.
Multiple sequence alignment (MSA) is the basis for a wide range of comparative sequence analyses from molecular phylogenetics to 3D structure prediction. Sophisticated algorithms have been developed for sequence alignment, but in practice, many errors can be expected and extensive portions of the MSA are unreliable. Hence, it is imperative to understand and characterize the various sources of errors in MSAs and to quantify site-specific alignment confidence. In this paper, we show that uncertainties in the guide tree used by progressive alignment methods are a major source of alignment uncertainty. We use this insight to develop a novel method for quantifying the robustness of each alignment column to guide tree uncertainty. We build on the widely used bootstrap method for perturbing the phylogenetic tree. Specifically, we generate a collection of trees and use each as a guide tree in the alignment algorithm, thus producing a set of MSAs. We next test the consistency of every column of the MSA obtained from the unperturbed guide tree with respect to the set of MSAs. We name this measure the “GUIDe tree based AligNment ConfidencE” (GUIDANCE) score. Using the Benchmark Alignment data BASE benchmark as well as simulation studies, we show that GUIDANCE scores accurately identify errors in MSAs. Additionally, we compare our results with the previously published Heads-or-Tails score and show that the GUIDANCE score is a better predictor of unreliably aligned regions.
PMCID: PMC2908709  PMID: 20207713
multiple sequence alignment; guide tree; phylogeny; bootstrap; alignment confidence
17.  PASS2 version 4: An update to the database of structure-based sequence alignments of structural domain superfamilies 
Nucleic Acids Research  2011;40(D1):D531-D534.
Accurate structure-based sequence alignments of distantly related proteins are crucial in gaining insight about protein domains that belong to a superfamily. The PASS2 database provides alignments of proteins related at the superfamily level and are characterized by low sequence identity. We thus report an automated, updated version of the superfamily alignment database known as PASS2.4, consisting of 1961 superfamilies and 10 569 protein domains, which is in direct correspondence with SCOP (1.75) database. Database organization, improved methods for efficient structure-based sequence alignments and the analysis of extreme distantly related proteins within superfamilies formed the focus of this update. Alignment of family-specific functional residues can be realized using such alignments and is shown using one superfamily as an example. The database of alignments and other related features can be accessed at
PMCID: PMC3245109  PMID: 22123743
18.  Using Structure to Explore the Sequence Alignment Space of Remote Homologs 
PLoS Computational Biology  2011;7(10):e1002175.
Protein structure modeling by homology requires an accurate sequence alignment between the query protein and its structural template. However, sequence alignment methods based on dynamic programming (DP) are typically unable to generate accurate alignments for remote sequence homologs, thus limiting the applicability of modeling methods. A central problem is that the alignment that is “optimal” in terms of the DP score does not necessarily correspond to the alignment that produces the most accurate structural model. That is, the correct alignment based on structural superposition will generally have a lower score than the optimal alignment obtained from sequence. Variations of the DP algorithm have been developed that generate alternative alignments that are “suboptimal” in terms of the DP score, but these still encounter difficulties in detecting the correct structural alignment. We present here a new alternative sequence alignment method that relies heavily on the structure of the template. By initially aligning the query sequence to individual fragments in secondary structure elements and combining high-scoring fragments that pass basic tests for “modelability”, we can generate accurate alignments within a small ensemble. Our results suggest that the set of sequences that can currently be modeled by homology can be greatly extended.
Author Summary
It has been suggested that, for nearly every protein sequence, there is already a protein with a similar structure in current protein structure databases. However, with poor or undetectable sequence relationships, it is expected that accurate alignments and models cannot be generated. Here we show that this is not the case, and that whenever structural relationship exists, there are usually local sequence relationships that can be used to generate an accurate alignment, no matter what the global sequence identity. However, this requires an alternative to the traditional dynamic programming algorithm and the consideration of a small ensemble of alignments. We present an algorithm, S4, and demonstrate that it is capable of generating accurate alignments in nearly all cases where a structural relationship exists between two proteins. Our results thus constitute an important advance in the full exploitation of the information in structural databases. That is, the expectation of an accurate alignment suggests that a meaningful model can be generated for nearly every sequence for which a suitable template exists.
PMCID: PMC3188491  PMID: 21998567
19.  PSI-BLAST-ISS: an intermediate sequence search tool for estimation of the position-specific alignment reliability 
BMC Bioinformatics  2005;6:185.
Protein sequence alignments have become indispensable for virtually any evolutionary, structural or functional study involving proteins. Modern sequence search and comparison methods combined with rapidly increasing sequence data often can reliably match even distantly related proteins that share little sequence similarity. However, even highly significant matches generally may have incorrectly aligned regions. Therefore when exact residue correspondence is used to transfer biological information from one aligned sequence to another, it is critical to know which alignment regions are reliable and which may contain alignment errors.
PSI-BLAST-ISS is a standalone Unix-based tool designed to delineate reliable regions of sequence alignments as well as to suggest potential variants in unreliable regions. The region-specific reliability is assessed by producing multiple sequence alignments in different sequence contexts followed by the analysis of the consistency of alignment variants. The PSI-BLAST-ISS output enables the user to simultaneously analyze alignment reliability between query and multiple homologous sequences. In addition, PSI-BLAST-ISS can be used to detect distantly related homologous proteins. The software is freely available at: .
PSI-BLAST-ISS is an effective reliability assessment tool that can be useful in applications such as comparative modelling or analysis of individual sequence regions. It favorably compares with the existing similar software both in the performance and functional features.
PMCID: PMC1187875  PMID: 16033659
20.  State of the art: refinement of multiple sequence alignments 
BMC Bioinformatics  2006;7:499.
Accurate multiple sequence alignments of proteins are very important in computational biology today. Despite the numerous efforts made in this field, all alignment strategies have certain shortcomings resulting in alignments that are not always correct. Refinement of existing alignment can prove to be an intelligent choice considering the increasing importance of high quality alignments in large scale high-throughput analysis.
We provide an extensive comparison of the performance of the alignment refinement algorithms. The accuracy and efficiency of the refinement programs are compared using the 3D structure-based alignments in the BAliBASE benchmark database as well as manually curated high quality alignments from Conserved Domain Database (CDD).
Comparison of performance for refined alignments revealed that despite the absence of dramatic improvements, our refinement method, REFINER, which uses conserved regions as constraints performs better in improving the alignments generated by different alignment algorithms. In most cases REFINER produces a higher-scoring, modestly improved alignment that does not deteriorate the well-conserved regions of the original alignment.
PMCID: PMC1654193  PMID: 17105653
21.  Improving the Alignment Quality of Consistency Based Aligners with an Evaluation Function Using Synonymous Protein Words 
PLoS ONE  2011;6(12):e27872.
Most sequence alignment tools can successfully align protein sequences with higher levels of sequence identity. The accuracy of corresponding structure alignment, however, decreases rapidly when considering distantly related sequences (<20% identity). In this range of identity, alignments optimized so as to maximize sequence similarity are often inaccurate from a structural point of view. Over the last two decades, most multiple protein aligners have been optimized for their capacity to reproduce structure-based alignments while using sequence information. Methods currently available differ essentially in the similarity measurement between aligned residues using substitution matrices, Fourier transform, sophisticated profile-profile functions, or consistency-based approaches, more recently.
In this paper, we present a flexible similarity measure for residue pairs to improve the quality of protein sequence alignment. Our approach, called SymAlign, relies on the identification of conserved words found across a sizeable fraction of the considered dataset, and supported by evolutionary analysis. These words are then used to define a position specific substitution matrix that better reflects the biological significance of local similarity. The experiment results show that the SymAlign scoring scheme can be incorporated within T-Coffee to improve sequence alignment accuracy. We also demonstrate that SymAlign is less sensitive to the presence of structurally non-similar proteins. In the analysis of the relationship between sequence identity and structure similarity, SymAlign can better differentiate structurally similar proteins from non- similar proteins.
We show that protein sequence alignments can be significantly improved using a similarity estimation based on weighted n-grams. In our analysis of the alignments thus produced, sequence conservation becomes a better indicator of structural similarity. SymAlign also provides alignment visualization that can display sub-optimal alignments on dot-matrices. The visualization makes it easy to identify well-supported alternative alignments that may not have been identified by dynamic programming. SymAlign is available at
PMCID: PMC3229492  PMID: 22163274
22.  Accurate statistical model of comparison between multiple sequence alignments 
Nucleic Acids Research  2008;36(7):2240-2248.
Comparison of multiple protein sequence alignments (MSA) reveals unexpected evolutionary relations between protein families and leads to exciting predictions of spatial structure and function. The power of MSA comparison critically depends on the quality of statistical model used to rank the similarities found in a database search, so that biologically relevant relationships are discriminated from spurious connections. Here, we develop an accurate statistical description of MSA comparison that does not originate from conventional models of single sequence comparison and captures essential features of protein families. As a final result, we compute E-values for the similarity between any two MSA using a mathematical function that depends on MSA lengths and sequence diversity. To develop these estimates of statistical significance, we first establish a procedure for generating realistic alignment decoys that reproduce natural patterns of sequence conservation dictated by protein secondary structure. Second, since similarity scores between these alignments do not follow the classic Gumbel extreme value distribution, we propose a novel distribution that yields statistically perfect agreement with the data. Third, we apply this random model to database searches and show that it surpasses conventional models in the accuracy of detecting remote protein similarities.
PMCID: PMC2367703  PMID: 18285364
23.  CORAL: aligning conserved core regions across domain families 
Bioinformatics  2009;25(15):1862-1868.
Motivation: Homologous protein families share highly conserved sequence and structure regions that are frequent targets for comparative analysis of related proteins and families. Many protein families, such as the curated domain families in the Conserved Domain Database (CDD), exhibit similar structural cores. To improve accuracy in aligning such protein families, we propose a profile–profile method CORAL that aligns individual core regions as gap-free units.
Results: CORAL computes optimal local alignment of two profiles with heuristics to preserve continuity within core regions. We benchmarked its performance on curated domains in CDD, which have pre-defined core regions, against COMPASS, HHalign and PSI-BLAST, using structure superpositions and comprehensive curator-optimized alignments as standards of truth. CORAL improves alignment accuracy on core regions over general profile methods, returning a balanced score of 0.57 for over 80% of all domain families in CDD, compared with the highest balanced score of 0.45 from other methods. Further, CORAL provides E-values to aid in detecting homologous protein families and, by respecting block boundaries, produces alignments with improved ‘readability’ that facilitate manual refinement.
Availability: CORAL will be included in future versions of the NCBI Cn3D/CDTree software, which can be downloaded at
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2712342  PMID: 19470584
24.  CentroidAlign-Web: A Fast and Accurate Multiple Aligner for Long Non-Coding RNAs 
Due to the recent discovery of non-coding RNAs (ncRNAs), multiple sequence alignment (MSA) of those long RNA sequences is becoming increasingly important for classifying and determining the functional motifs in RNAs. However, not only primary (nucleotide) sequences, but also secondary structures of ncRNAs are closely related to their function and are conserved evolutionarily. Hence, information about secondary structures should be considered in the sequence alignment of ncRNAs. Yet, in general, a huge computational time is required in order to compute MSAs, taking secondary structure information into account. In this paper, we describe a fast and accurate web server, called CentroidAlign-Web, which can handle long RNA sequences. The web server also appropriately incorporates information about known secondary structures into MSAs. Computational experiments indicate that our web server is fast and accurate enough to handle long RNA sequences. CentroidAlign-Web is freely available from
PMCID: PMC3634467  PMID: 23507751
ncRNAs; rRNAs; multiple sequence alignment (MSA); structural alignment; secondary structure; consensus structures; web server
25.  Sequence Alignment by Cross-Correlation 
Many recent advances in biology and medicine have resulted from DNA sequence alignment algorithms and technology. Traditional approaches for the matching of DNA sequences are based either on global alignment schemes or heuristic schemes that seek to approximate global alignment algorithms while providing higher computational efficiency. This report describes an approach using the mathematical operation of cross-correlation to compare sequences. It can be implemented using the fast fourier transform for computational efficiency. The algorithm is summarized and sample applications are given. These include gene sequence alignment in long stretches of genomic DNA, finding sequence similarity in distantly related organisms, demonstrating sequence similarity in the presence of massive (approximately 90%) random point mutations, comparing sequences related by internal rearrangements (tandem repeats) within a gene, and investigating fusion proteins. Application to RNA and protein sequence alignment is also discussed. The method is efficient, sensitive, and robust, being able to find sequence similarities where other alignment algorithms may perform poorly.
PMCID: PMC2291754  PMID: 16522868
Sequence alignment; algorithm; software; cross-correlation

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