Summary: Iterative similarity searches with PSI-BLAST position-specific score matrices (PSSMs) find many more homologs than single searches, but PSSMs can be contaminated when homologous alignments are extended into unrelated protein domains—homologous over-extension (HOE). PSI-Search combines an optimal Smith–Waterman local alignment sequence search, using SSEARCH, with the PSI-BLAST profile construction strategy. An optional sequence boundary-masking procedure, which prevents alignments from being extended after they are initially included, can reduce HOE errors in the PSSM profile. Preventing HOE improves selectivity for both PSI-BLAST and PSI-Search, but PSI-Search has ~4-fold better selectivity than PSI-BLAST and similar sensitivity at 50% and 60% family coverage. PSI-Search is also produces 2- for 4-fold fewer false-positives than JackHMMER, but is ~5% less sensitive.
Availability and implementation: PSI-Search is available from the authors as a standalone implementation written in Perl for Linux-compatible platforms. It is also available through a web interface (www.ebi.ac.uk/Tools/sss/psisearch) and SOAP and REST Web Services (www.ebi.ac.uk/Tools/webservices).
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSI-BLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.
All-versus-all BLAST, which searches for homologous pairs of sequences in a database of proteins, is used to identify potential orthologs, to find new protein families, and to provide rapid access to these homology relationships. As DNA sequencing accelerates and data sets grow, all-versus-all BLAST has become computationally demanding.
We present FastBLAST, a heuristic replacement for all-versus-all BLAST that relies on alignments of proteins to known families, obtained from tools such as PSI-BLAST and HMMer. FastBLAST avoids most of the work of all-versus-all BLAST by taking advantage of these alignments and by clustering similar sequences. FastBLAST runs in two stages: the first stage identifies additional families and aligns them, and the second stage quickly identifies the homologs of a query sequence, based on the alignments of the families, before generating pairwise alignments. On 6.53 million proteins from the non-redundant Genbank database (“NR”), FastBLAST identifies new families 25 times faster than all-versus-all BLAST. Once the first stage is completed, FastBLAST identifies homologs for the average query in less than 5 seconds (8.6 times faster than BLAST) and gives nearly identical results. For hits above 70 bits, FastBLAST identifies 98% of the top 3,250 hits per query.
FastBLAST enables research groups that do not have supercomputers to analyze large protein sequence data sets. FastBLAST is open source software and is available at http://microbesonline.org/fastblast.
Position specific score matrices (PSSMs) are derived from multiple sequence alignments to aid in the recognition of distant protein sequence relationships. The PSI-BLAST protein database search program derives the column scores of its PSSMs with the aid of pseudocounts, added to the observed amino acid counts in a multiple alignment column. In the absence of theory, the number of pseudocounts used has been a completely empirical parameter. This article argues that the minimum description length principle can motivate the choice of this parameter. Specifically, for realistic alignments, the principle supports the practice of using a number of pseudocounts essentially independent of alignment size. However, it also implies that more highly conserved columns should use fewer pseudocounts, increasing the inter-column contrast of the implied PSSMs. A new method for calculating pseudocounts that significantly improves PSI-BLAST's; retrieval accuracy is now employed by default.
We have characterized a novel type of PSI-BLAST error, homologous over-extension (HOE), using embedded PFAM domain queries on searches against a reference library containing Pfam-annotated UniProt sequences and random synthetic sequences. PSI-BLAST makes two types of errors: alignments to non-homologous regions and HOE alignments that begin in a homologous region, but extend beyond the homology into neighboring sequence regions. When the neighboring sequence region contains a non-homologous domain, PSI-BLAST can incorporate the unrelated sequence into its position specific scoring matrix, which then finds non-homologous proteins with significant expectation values. HOE accounts for the largest fraction of the initial false positive (FP) errors, and the largest fraction of FPs at iteration 5. In searches against complete protein sequences, 5–9% of alignments at iteration 5 are non-homologous. HOE frequently begins in a partial protein domain; when partial domains are removed from the library, HOE errors decrease from 16 to 3% of weighted coverage (hard queries; 35–5% for sampled queries) and no-error searches increase from 2 to 58% weighed coverage (hard; 16–78% sampled). When HOE is reduced by not extending previously found sequences, PSI-BLAST specificity improves 4–8-fold, with little loss in sensitivity.
In biological sequence analysis, position specific scoring matrices (PSSMs) are widely used to represent sequence motifs in nucleotide as well as amino acid sequences. Searching with PSSMs in complete genomes or large sequence databases is a common, but computationally expensive task.
We present a new non-heuristic algorithm, called ESAsearch, to efficiently find matches of PSSMs in large databases. Our approach preprocesses the search space, e.g., a complete genome or a set of protein sequences, and builds an enhanced suffix array that is stored on file. This allows the searching of a database with a PSSM in sublinear expected time. Since ESAsearch benefits from small alphabets, we present a variant operating on sequences recoded according to a reduced alphabet. We also address the problem of non-comparable PSSM-scores by developing a method which allows the efficient computation of a matrix similarity threshold for a PSSM, given an E-value or a p-value. Our method is based on dynamic programming and, in contrast to other methods, it employs lazy evaluation of the dynamic programming matrix. We evaluated algorithm ESAsearch with nucleotide PSSMs and with amino acid PSSMs. Compared to the best previous methods, ESAsearch shows speedups of a factor between 17 and 275 for nucleotide PSSMs, and speedups up to factor 1.8 for amino acid PSSMs. Comparisons with the most widely used programs even show speedups by a factor of at least 3.8. Alphabet reduction yields an additional speedup factor of 2 on amino acid sequences compared to results achieved with the 20 symbol standard alphabet. The lazy evaluation method is also much faster than previous methods, with speedups of a factor between 3 and 330.
Our analysis of ESAsearch reveals sublinear runtime in the expected case, and linear runtime in the worst case for sequences not shorter than |A
MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBamrtHrhAL1wy0L2yHvtyaeHbnfgDOvwBHrxAJfwnaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaWaaeGaeaaakeaaimaacqWFaeFqaaa@3821@|m + m - 1, where m is the length of the PSSM and A
MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBamrtHrhAL1wy0L2yHvtyaeHbnfgDOvwBHrxAJfwnaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaWaaeGaeaaakeaaimaacqWFaeFqaaa@3821@ a finite alphabet. In practice, ESAsearch shows superior performance over the most widely used programs, especially for DNA sequences. The new algorithm for accurate on-the-fly calculations of thresholds has the potential to replace formerly used approximation approaches. Beyond the algorithmic contributions, we provide a robust, well documented, and easy to use software package, implementing the ideas and algorithms presented in this manuscript.
PSI-BLAST is an iterative program to search a database for proteins
with distant similarity to a query sequence. We investigated over
a dozen modifications to the methods used in PSI-BLAST, with the goal
of improving accuracy in finding true positive matches. To evaluate
performance we used a set of 103 queries for which the true positives
in yeast had been annotated by human experts, and a popular measure
of retrieval accuracy (ROC) that can be normalized to take on values
between 0 (worst) and 1 (best). The modifications we consider novel
improve the ROC score from 0.758 ± 0.005
to 0.895 ± 0.003. This does not include
the benefits from four modifications we included in the ‘baseline’ version,
even though they were not implemented in PSI-BLAST version 2.0.
The improvement in accuracy was confirmed on a small second test
set. This test involved analyzing three protein families with curated
lists of true positives from the non-redundant protein database.
The modification that accounts for the majority of the improvement
is the use, for each database sequence, of a position-specific scoring
system tuned to that sequence’s amino acid composition.
The use of composition-based statistics is particularly beneficial
for large-scale automated applications of PSI-BLAST.
Many DNA regulatory elements occur as multiple instances within a target promoter. Gibbs sampling programs for finding DNA regulatory elements de novo can be prohibitively slow in locating all instances of such an element in a sequence set.
We describe an improvement to the A-GLAM computer program, which predicts regulatory elements within DNA sequences with Gibbs sampling. The improvement adds an optional "scanning step" after Gibbs sampling. Gibbs sampling produces a position specific scoring matrix (PSSM). The new scanning step resembles an iterative PSI-BLAST search based on the PSSM. First, it assigns an "individual score" to each subsequence of appropriate length within the input sequences using the initial PSSM. Second, it computes an E-value from each individual score, to assess the agreement between the corresponding subsequence and the PSSM. Third, it permits subsequences with E-values falling below a threshold to contribute to the underlying PSSM, which is then updated using the Bayesian calculus. A-GLAM iterates its scanning step to convergence, at which point no new subsequences contribute to the PSSM. After convergence, A-GLAM reports predicted regulatory elements within each sequence in order of increasing E-values, so users have a statistical evaluation of the predicted elements in a convenient presentation. Thus, although the Gibbs sampling step in A-GLAM finds at most one regulatory element per input sequence, the scanning step can now rapidly locate further instances of the element in each sequence.
Datasets from experiments determining the binding sites of transcription factors were used to evaluate the improvement to A-GLAM. Typically, the datasets included several sequences containing multiple instances of a regulatory motif. The improvements to A-GLAM permitted it to predict the multiple instances.
SCANPS performs iterative profile searching similar to PSI-BLAST but with full dynamic programing on each cycle and on-the-fly estimation of significance. This combination gives good sensitivity and selectivity that outperforms PSI-BLAST in domain-searching benchmarks. Although computationally expensive, SCANPS exploits onchip parallelism (MMX and SSE2 instructions on Intel chips) as well as MPI parallelism to give acceptable turnround times even for large databases. A web server developed to run SCANPS searches is now available at http://www.compbio.dundee.ac.uk/www-scanps. The server interface allows a range of different protein sequence databases to be searched including the SCOP database of protein domains. The server provides the user with regularly updated versions of the main protein sequence databases and is backed up by significant computing resources which ensure that searches are performed rapidly. For SCOP searches, the results may be viewed in a new tree-based representation that reflects the structure of the SCOP hierarchy; this aids the user in placing each hit in the context of its SCOP classification and understanding its relationship to other domains in SCOP.
A SIB-BLAST web server (http://sib-blast.osc.edu) has been established for investigators to use the SimpleIsBeautiful (SIB) algorithm for sequence-based homology detection. SIB was developed to overcome the model corruption frequently observed in the later iterations of PSI-BLAST searches. The algorithm compares resultant hits from the second iteration to the final iteration of a PSI-BLAST search, calculates the figure of merit for each ‘overlapped’ hit and re-ranks the hits according to their figure of merit. By validating hits generated from the last profile against hits from the first profile when the model is least corrupted, the true and false positives are better delineated, which in turn, improves the accuracy of iterative PSI-BLAST searches. Notably, this improvement to PSI-BLAST comes at minimal computational cost as SIB-BLAST utilizes existing results already produced in a PSI-BLAST search.
Summary: A variety of specialist databases have been developed to facilitate the study of allergens. However, these databases either contain different subsets of allergen data or are deficient in tools for assessing potential allergenicity of proteins. Here, we describe Allergen Atlas, a comprehensive repository of experimentally validated allergen sequences collected from in-house laboratory, online data submission, literature reports and all existing general-purpose and specialist databases. Each entry was manually verified, classified and hyperlinked to major databases including Swiss-Prot, Protein Data Bank (PDB), Gene Ontology (GO), Pfam and PubMed. The database is integrated with analysis tools that include: (i) keyword search, (ii) BLAST, (iii) position-specific iterative BLAST (PSI-BLAST), (iv) FAO/WHO criteria search, (v) graphical representation of allergen information network and (vi) online data submission. The latest version contains information of 1593 allergen sequences (496 IUIS allergens, 978 experimentally verified allergens and 119 new sequences), 56 IgE epitope sequences, 679 links to PDB structures and 155 links to Pfam domains.
Availability: Allergen Atlas is freely available at http://tiger.dbs.nus.edu.sg/ATLAS/.
The Conserved Domain Database (CDD) is now indexed as a separate database within the Entrez system and linked to other Entrez databases such as MEDLINE®. This allows users to search for domain types by name, for example, or to view the domain architecture of any protein in Entrez's sequence database. CDD can be accessed on the WorldWideWeb at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. Users may also employ the CD-Search service to identify conserved domains in new sequences, at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. CD-Search results, and pre-computed links from Entrez's protein database, are calculated using the RPS-BLAST algorithm and Position Specific Score Matrices (PSSMs) derived from CDD alignments. CD-Searches are also run by default for protein–protein queries submitted to BLAST® at http://www.ncbi.nlm.nih.gov/BLAST.
CDD mirrors the publicly available domain alignment collections SMART and PFAM, and now also contains alignment models curated at NCBI. Structure information is used to identify the core substructure likely to be present in all family members, and to produce sequence alignments consistent with structure conservation. This alignment model allows NCBI curators to annotate ‘columns’ corresponding to functional sites conserved among family members.
Motivation: The BLAST software package for sequence comparison speeds up homology search by preprocessing a query sequence into a lookup table. Numerous research studies have suggested that preprocessing the database instead would give better performance. However, production usage of sequence comparison methods that preprocess the database has been limited to programs such as BLAT and SSAHA that are designed to find matches when query and database subsequences are highly similar.
Results: We developed a new version of the MegaBLAST module of BLAST that does the initial phase of finding short seeds for matches by searching a database index. We also developed a program makembindex that preprocesses the database into a data structure for rapid seed searching. We show that the new ‘indexed MegaBLAST’ is faster than the ‘non-indexed’ version for most practical uses. We show that indexed MegaBLAST is faster than miBLAST, another implementation of BLAST nucleotide searching with a preprocessed database, for most of the 200 queries we tested. To deploy indexed MegaBLAST as part of NCBI'sWeb BLAST service, the storage of databases and the queueing mechanism were modified, so that some machines are now dedicated to serving queries for a specific database. The response time for such Web queries is now faster than it was when each computer handled queries for multiple databases.
Availability: The code for indexed MegaBLAST is part of the blastn program in the NCBI C++ toolkit. The preprocessor program makembindex is also in the toolkit. Indexed MegaBLAST has been used in production on NCBI's Web BLAST service to search one version of the human and mouse genomes since October 2007. The Linux command-line executables for blastn and makembindex, documentation, and some query sets used to carry out the tests described below are available in the directory: ftp://ftp.ncbi.nlm.nih.gov/pub/agarwala/indexed_megablast
Supplementary information: Supplementary data are available at Bioinformatics online.
Prediction of bacterial virulent protein sequences has implications for identification and characterization of novel virulence-associated factors, finding novel drug/vaccine targets against proteins indispensable to pathogenicity, and understanding the complex virulence mechanism in pathogens.
In the present study we propose a bacterial virulent protein prediction method based on bi-layer cascade Support Vector Machine (SVM). The first layer SVM classifiers were trained and optimized with different individual protein sequence features like amino acid composition, dipeptide composition (occurrences of the possible pairs of ith and i+1th amino acid residues), higher order dipeptide composition (pairs of ith and i+2nd residues) and Position Specific Iterated BLAST (PSI-BLAST) generated Position Specific Scoring Matrices (PSSM). In addition, a similarity-search based module was also developed using a dataset of virulent and non-virulent proteins as BLAST database. A five-fold cross-validation technique was used for the evaluation of various prediction strategies in this study. The results from the first layer (SVM scores and PSI-BLAST result) were cascaded to the second layer SVM classifier to train and generate the final classifier. The cascade SVM classifier was able to accomplish an accuracy of 81.8%, covering 86% area in the Receiver Operator Characteristic (ROC) plot, better than that of either of the layer one SVM classifiers based on single or multiple sequence features.
VirulentPred is a SVM based method to predict bacterial virulent proteins sequences, which can be used to screen virulent proteins in proteomes. Together with experimentally verified virulent proteins, several putative, non annotated and hypothetical protein sequences have been predicted to be high scoring virulent proteins by the prediction method. VirulentPred is available as a freely accessible World Wide Web server – VirulentPred, at .
Annotating the tremendous amount of sequence information being generated requires accurate automated methods for recognizing homology. Although sequence similarity is only one of many indicators of evolutionary homology, it is often the only one used. Here we find that supplementing sequence similarity with information from biomedical literature is successful in increasing the accuracy of homology search results. We modified the PSI-BLAST algorithm to use literature similarity in each iteration of its database search. The modified algorithm is evaluated and compared to standard PSI-BLAST in searching for homologous proteins. The performance of the modified algorithm achieved 32% recall with 95% precision, while the original one achieved 33% recall with 84% precision; the literature similarity requirement preserved the sensitive characteristic of the PSI-BLAST algorithm while improving the precision.
Understanding how biomolecules interact is a major task of systems biology. To model protein-nucleic acid interactions, it is important to identify the DNA or RNA-binding residues in proteins. Protein sequence features, including the biochemical property of amino acids and evolutionary information in terms of position-specific scoring matrix (PSSM), have been used for DNA or RNA-binding site prediction. However, PSSM is rather designed for PSI-BLAST searches, and it may not contain all the evolutionary information for modelling DNA or RNA-binding sites in protein sequences.
In the present study, several new descriptors of evolutionary information have been developed and evaluated for sequence-based prediction of DNA and RNA-binding residues using support vector machines (SVMs). The new descriptors were shown to improve classifier performance. Interestingly, the best classifiers were obtained by combining the new descriptors and PSSM, suggesting that they captured different aspects of evolutionary information for DNA and RNA-binding site prediction. The SVM classifiers achieved 77.3% sensitivity and 79.3% specificity for prediction of DNA-binding residues, and 71.6% sensitivity and 78.7% specificity for RNA-binding site prediction.
Predictions at this level of accuracy may provide useful information for modelling protein-nucleic acid interactions in systems biology studies. We have thus developed a web-based tool called BindN+ (http://bioinfo.ggc.org/bindn+/) to make the SVM classifiers accessible to the research community.
Motivation: A typical PSI-BLAST search consists of iterative scanning and alignment of a large sequence database during which a scoring profile is progressively built and refined. Such a profile can also be stored and used to search against a different database of sequences. Using it to search against a database of consensus rather than native sequences is a simple add-on that boosts performance surprisingly well. The improvement comes at a price: we hypothesized that random alignment score statistics would differ between native and consensus sequences. Thus PSI-BLAST-based profile searches against consensus sequences might incorrectly estimate statistical significance of alignment scores. In addition, iterative searches against consensus databases may fail. Here, we addressed these challenges in an attempt to harness the full power of the combination of PSI-BLAST and consensus sequences.
Results: We studied alignment score statistics for various types of consensus sequences. In general, the score distribution parameters of profile-based consensus sequence alignments differed significantly from those derived for the native sequences. PSI-BLAST partially compensated for the parameter variation. We have identified a protocol for building specialized consensus sequences that significantly improved search sensitivity and preserved score distribution parameters. As a result, PSI-BLAST profiles can be used to search specialized consensus sequences without sacrificing estimates of statistical significance. We also provided results indicating that iterative PSI-BLAST searches against consensus sequences could work very well. Overall, we showed how a very popular and effective method could be used to identify significantly more relevant similarities among protein sequences.
A typical PSI-BLAST search consists of iterative scanning and alignment of a large sequence database during which a scoring profile is progressively built and refined. Such a profile can also be stored and used to search against a different database of sequences. Using it to search against a database of consensus rather than native sequences is a simple add-on that boosts performance surprisingly well. The improvement comes at a price: we hypothesized that random alignment score statistics would differ between native and consensus sequences. Thus PSI-BLAST-based profile searches against consensus sequences might incorrectly estimate statistical significance of alignment scores. In addition, iterative searches against consensus databases may fail. Here, we addressed these challenges in an attempt to harness the full power of the combination of PSI-BLAST and consensus sequences.
We studied alignment score statistics for various types of consensus sequences. In general, the score distribution parameters of profile-based consensus sequence alignments differed significantly from those derived for the native sequences. PSI-BLAST partially compensated for the parameter variation. We have identified a protocol for building specialized consensus sequences that significantly improved search sensitivity and preserved score distribution parameters. As a result, PSI-BLAST profiles can be used to search specialized consensus sequences without sacrificing estimates of statistical significance. We also provided results indicating that iterative PSI-BLAST searches against consensus sequences could work very well. Overall, we showed how a widely popular and effective method could be used to identify significantly more relevant similarities among protein sequences.
Sequence similarity searching is a powerful tool to help develop hypotheses in the quest to assign functional, structural and evolutionary information to DNA and protein sequences. As sequence databases continue to grow exponentially, it becomes increasingly important to repeat searches at frequent intervals, and similarity searches retrieve larger and larger sets of results. New and potentially significant results may be buried in a long list of previously obtained sequence hits from past searches.
ReHAB (Recent Hits Acquired from BLAST) is a tool for finding new protein hits in repeated PSI-BLAST searches. ReHAB compares results from PSI-BLAST searches performed with two versions of a protein sequence database and highlights hits that are present only in the updated database. Results are presented in an easily comprehended table, or in a BLAST-like report, using colors to highlight the new hits. ReHAB is designed to handle large numbers of query sequences, such as whole genomes or sets of genomes. Advanced computer skills are not needed to use ReHAB; the graphics interface is simple to use and was designed with the bench biologist in mind.
This software greatly simplifies the problem of evaluating the output of large numbers of protein database searches.
Template selection and target-template alignment are critical steps for template-based modeling (TBM) methods. To identify the template for the twilight zone of 15~25% sequence similarity between targets and templates is still difficulty for template-based protein structure prediction. This study presents the (PS)2-v2 server, based on our original server with numerous enhancements and modifications, to improve reliability and applicability.
To detect homologous proteins with remote similarity, the (PS)2-v2 server utilizes the S2A2 matrix, which is a 60 × 60 substitution matrix using the secondary structure propensities of 20 amino acids, and the position-specific sequence profile (PSSM) generated by PSI-BLAST. In addition, our server uses multiple templates and multiple models to build and assess models. Our method was evaluated on the Lindahl benchmark for fold recognition and ProSup benchmark for sequence alignment. Evaluation results indicated that our method outperforms sequence-profile approaches, and had comparable performance to that of structure-based methods on these benchmarks. Finally, we tested our method using the 154 TBM targets of the CASP8 (Critical Assessment of Techniques for Protein Structure Prediction) dataset. Experimental results show that (PS)2-v2 is ranked 6th among 72 severs and is faster than the top-rank five serves, which utilize ab initio methods.
Experimental results demonstrate that (PS)2-v2 with the S2A2 matrix is useful for template selections and target-template alignments by blending the amino acid and structural propensities. The multiple-template and multiple-model strategies are able to significantly improve the accuracies for target-template alignments in the twilight zone. We believe that this server is useful in structure prediction and modeling, especially in detecting homologous templates with sequence similarity in the twilight zone.
The large numbers of protein sequences generated by whole genome sequencing projects require rapid and accurate methods of annotation. The detection of homology through computational sequence analysis is a powerful tool in determining the complex evolutionary and functional relationships that exist between proteins. Homology search algorithms employ amino acid substitution matrices to detect similarity between proteins sequences. The substitution matrices in common use today are constructed using sequences aligned without reference to protein structure. Here we present amino acid substitution matrices constructed from the alignment of a large number of protein domain structures from the structural classification of proteins (SCOP) database. We show that when incorporated into the homology search algorithms BLAST and PSI-blast, the structure-based substitution matrices enhance the efficacy of detecting remote homologs.
computational biology; protein homology; amino acid substitution matrix; protein structure
BLAST searches are widely used for sequence alignment. The search results are commonly adopted for various functional and comparative genomics tasks such as annotating unknown sequences, investigating gene models and comparing two sequence sets. Advances in sequencing technologies pose challenges for high-throughput analysis of large-scale sequence data. A number of programs and hardware solutions exist for efficient BLAST searching, but there is a lack of generic software solutions for mining and personalized management of the results. Systematically reviewing the results and identifying information of interest remains tedious and time-consuming.
Personal BLAST Navigator (PLAN) is a versatile web platform that helps users to carry out various personalized pre- and post-BLAST tasks, including: (1) query and target sequence database management, (2) automated high-throughput BLAST searching, (3) indexing and searching of results, (4) filtering results online, (5) managing results of personal interest in favorite categories, (6) automated sequence annotation (such as NCBI NR and ontology-based annotation). PLAN integrates, by default, the Decypher hardware-based BLAST solution provided by Active Motif Inc. with a greatly improved efficiency over conventional BLAST software. BLAST results are visualized by spreadsheets and graphs and are full-text searchable. BLAST results and sequence annotations can be exported, in part or in full, in various formats including Microsoft Excel and FASTA. Sequences and BLAST results are organized in projects, the data publication levels of which are controlled by the registered project owners. In addition, all analytical functions are provided to public users without registration.
PLAN has proved a valuable addition to the community for automated high-throughput BLAST searches, and, more importantly, for knowledge discovery, management and sharing based on sequence alignment results. The PLAN web interface is platform-independent, easily configurable and capable of comprehensive expansion, and user-intuitive. PLAN is freely available to academic users at . The source code for local deployment is provided under free license. Full support on system utilization, installation, configuration and customization are provided to academic users.
Sequence similarity searching is a very important bioinformatics task. While Basic Local Alignment Search Tool (BLAST) outperforms exact methods through its use of heuristics, the speed of the current BLAST software is suboptimal for very long queries or database sequences. There are also some shortcomings in the user-interface of the current command-line applications.
We describe features and improvements of rewritten BLAST software and introduce new command-line applications. Long query sequences are broken into chunks for processing, in some cases leading to dramatically shorter run times. For long database sequences, it is possible to retrieve only the relevant parts of the sequence, reducing CPU time and memory usage for searches of short queries against databases of contigs or chromosomes. The program can now retrieve masking information for database sequences from the BLAST databases. A new modular software library can now access subject sequence data from arbitrary data sources. We introduce several new features, including strategy files that allow a user to save and reuse their favorite set of options. The strategy files can be uploaded to and downloaded from the NCBI BLAST web site.
The new BLAST command-line applications, compared to the current BLAST tools, demonstrate substantial speed improvements for long queries as well as chromosome length database sequences. We have also improved the user interface of the command-line applications.
Protein families often are characterized by conserved sequence patterns or motifs. A researcher frequently wishes to evaluate the significance of a specific pattern within a protein, or to exploit knowledge of known motifs to aid the recognition of greatly diverged but homologous family members. To assist in these efforts, the pattern-hit initiated BLAST (PHI-BLAST) program described here takes as input both a protein sequence and a pattern of interest that it contains. PHI-BLAST searches a protein database for other instances of the input pattern, and uses those found as seeds for the construction of local alignments to the query sequence. The random distribution of PHI-BLAST alignment scores is studied analytically and empirically. In many instances, the program is able to detect statistically significant similarity between homologous proteins that are not recognizably related using traditional single-pass database search methods. PHI-BLAST is applied to the analysis of CED4-like cell death regulators, HS90-type ATPase domains, archaeal tRNA nucleotidyltransferases and archaeal homologs of DnaG-type DNA primases.
Owing to high evolutionary divergence, it is not always possible to identify distantly related protein domains by sequence search techniques. Intermediate sequences possess sequence features of more than one protein and facilitate detection of remotely related proteins. We have demonstrated recently the employment of Cascade PSI-BLAST where we perform PSI-BLAST for many ‘generations’, initiating searches from new homologues as well. Such a rigorous propagation through generations of PSI-BLAST employs effectively the role of intermediates in detecting distant similarities between proteins. This approach has been tested on a large number of folds and its performance in detecting superfamily level relationships is ∼35% better than simple PSI-BLAST searches. We present a web server for this search method that permits users to perform Cascade PSI-BLAST searches against the Pfam, SCOP and SwissProt databases. The URL for this server is .