Gene structure prediction from consensus spliced alignment of multiple ESTs matching the same genomic locus

MOTIVATION: Accurate gene structure annotation is a challenging computational problem in genomics. The best results are achieved with spliced alignment of full-length cDNAs or multiple expressed sequence tags (ESTs) with sufficient overlap to cover the entire gene. For most species, cDNA and EST collections are far from comprehensive. We sought to overcome this bottleneck by exploring the possibility of using combined EST resources from fairly diverged species that still share a common gene space. Previous spliced alignment tools were found inadequate for this task because they rely on very high sequence similarity between the ESTs and the genomic DNA. RESULTS: We have developed a computer program, GeneSeqer, which is capable of aligning thousands of ESTs with a long genomic sequence in a reasonable amount of time. The algorithm is uniquely designed to tolerate a high percentage of mismatches and insertions or deletions in the EST relative to the genomic template. This feature allows use of non-cognate ESTs for gene structure prediction, including ESTs derived from duplicated genes and homologous genes from related species. The increased gene prediction sensitivity results in part from novel splice site prediction models that are also available as a stand-alone splice site prediction tool. We assessed GeneSeqer performance relative to a standard Arabidopsis thaliana gene set and demonstrate its utility for plant genome annotation. In particular, we propose that this method provides a timely tool for the annotation of the rice genome, using abundant ESTs from other cereals and plants. AVAILABILITY: The source code is available for download at http://bioinformatics.iastate.edu/bioinformatics2go/gs/download.html. Web servers for Arabidopsis and other plant species are accessible at http://www.plantgdb.org/cgi-bin/AtGeneSeqer.cgi and http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi, respectively. For non-plant species, use http://bioinformatics.iastate.edu/cgi-bin/gs.cgi. The splice site prediction tool (SplicePredictor) is distributed with the GeneSeqer code. A SplicePredictor web server is available at http://bioinformatics.iastate.edu/cgi-bin/sp.cgi     

Gene discovery using the maize genome database ZmDB

Zea mays DataBase (ZmDB) is a repository and analysis tool for sequence, expression and phenotype data of the major crop plant maize. The data accessible in ZmDB are mostly generated in a large collaborative project of maize gene discovery, sequencing and phenotypic analysis using a transposon tagging strategy and expressed sequence tag (EST) sequencing. ESTs constitute most of the current content. Database search tools, convenient links to external databases, and novel sequence analysis programs for spliced alignment are provided and together serve as an efficient protocol for gene discovery by sequence inspection. ZmDB can be accessed at http://zmdb. iastate.edu. ZmDB also provides web-based ordering of materials generated in the project, including EST and genomic DNA clones, seeds of mutant plants and microarrays of amplified EST and genomic DNA sequences.     

ZmDB,an integrated database for maize genome research

Zea mays DataBase (ZmDB) seeks to provide a comprehensive view of maize (corn) genetics by linking genomic sequence data with gene expression analysis and phenotypes of mutant plants. ZmDB originated in 1999 as the Web portal for a large project of maize gene discovery, sequencing and phenotypic analysis using a transposon tagging strategy and expressed sequence tag (EST) sequencing. Recently, ZmDB has broadened its scope to include all public maize ESTs, genome survey sequences (GSSs), and protein sequences. More than 170 000 ESTs are currently clustered into approximately 20 000 contigs and about an equal number of apparent singlets. These clusters are continuously updated and annotated with respect to potential encoded protein products. More than 100 000 GSSs are similarly assembled and annotated by spliced alignment with EST and protein sequences. The ZmDB interface provides quick access to analytical tools for further sequence analysis. Every sequence record is linked to several display options and similarity search tools, including services for multiple sequence alignment, protein domain determination and spliced alignment. Furthermore, ZmDB provides web-based ordering of materials generated in the project, including ESTs, ordered collections of genomic sequences tagged with the RescueMu transposon and microarrays of amplified ESTs. ZmDB can be accessed at http://zmdb.iastate.edu/.     

Gene structure prediction by spliced alignment of genomic DNA with protein sequences: increased accuracy by differential splice site scoring

Gene identification in genomic DNA from eukaryotes is complicated by the vast combinatorial possibilities of potential exon assemblies. If the gene encodes a protein that is closely related to known proteins, gene identification is aided by matching similarity of potential translation products to those target proteins. The genomic DNA and protein sequences can be aligned directly by scoring the implied residues of in-frame nucleotide triplets against the protein residues in conventional ways, while allowing for long gaps in the alignment corresponding to introns in the genomic DNA. We describe a novel method for such spliced alignment. The method derives an optimal alignment based on scoring for both sequence similarity of the predicted gene product to the protein sequence and intrinsic splice site strength of the predicted introns. Application of the method to a representative set of 50 known genes from Arabidopsis thaliana showed significant improvement in prediction accuracy compared to previous spliced alignment methods. The method is also more accurate than ab initio gene prediction methods, provided sufficiently close target proteins are available. In view of the fast growth of public sequence repositories, we argue that close targets will be available for the majority of novel genes, making spliced alignment an excellent practical tool for high-throughput automated genome annotation.     

Refined annotation of the Arabidopsis genome by complete expressed sequence tag mapping

Expressed sequence tags (ESTs) currently encompass more entries in the public databases than any other form of sequence data. Thus, EST data sets provide a vast resource for gene identification and expression profiling. We have mapped the complete set of 176,915 publicly available Arabidopsis EST sequences onto the Arabidopsis genome using GeneSeqer, a spliced alignment program incorporating sequence similarity and splice site scoring. About 96% of the available ESTs could be properly aligned with a genomic locus, with the remaining ESTs deriving from organelle genomes and non-Arabidopsis sources or displaying insufficient sequence quality for alignment. The mapping provides verified sets of EST clusters for evaluation of EST clustering programs. Analysis of the spliced alignments suggests corrections to current gene structure annotation and provides examples of alternative and non-canonical pre-mRNA splicing. All results of this study were parsed into a database and are accessible via a flexible Web interface at http://www.plantgdb.org/AtGDB/.     

Gene structure identification with MyGV using cDNA evidence and protein homologs to improve ab initio predictions

MyGV is an application to visualize (potentially genome-scale) gene structure annotation and prediction. The output of any external gene prediction program can be easily converted to a generalized format for input into MyGV. The application displays all input simultaneously in graphical representation, with a toggle option for a text-based view. Zooming capabilities allow detailed comparisons for specific genome locations. The tool is particularly helpful for refinement of ab initio predicted gene structures by spliced alignment with cDNA or protein homologs. AVAILABILITY: The program was written in Java and is freely available to non-commercial users by electronic download from http://bioinformatics.iastate.edu/bioinformatics2go/MyGV.     

The intronerator: exploring introns and alternative splicing in Caenorhabditis elegans

The Intronerator (http://www.cse.ucsc.edu/ approximately kent/intronerator/ ) is a set of web-based tools for exploring RNA splicing and gene structure in Caenorhabditis elegans. It includes a display of cDNA alignments with the genomic sequence, a catalog of alternatively spliced genes and a database of introns. The cDNA alignments include >100 000 ESTs and almost 1000 full-length cDNAs. ESTs from embryos and mixed stage animals as well as full-length cDNAs can be compared in the alignment display with each other and with predicted genes. The alt-splicing catalog includes 844 open reading frames for which there is evidence of alternative splicing of pre-mRNA. The intron database includes 28 478 introns, and can be searched for patterns near the splice junctions.     

GeneSeqer@PlantGDB: Gene structure prediction in plant genomes

The GeneSeqer@PlantGDB Web server (http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi) provides a gene structure prediction tool tailored for applications to plant genomic sequences. Predictions are based on spliced alignment with source-native ESTs and full-length cDNAs or non-native probes derived from putative homologous genes. The tool is illustrated with applications to refinement of current gene structure annotation and de novo annotation of draft genomic sequences. The service should facilitate expert annotation as a community effort by providing convenient access to all public plant sequences via the PlantGDB database, a simple four-step protocol for spliced alignment and visually appealing displays of the predicted gene structures in addition to detailed sequence alignments.     

Calign: aligning sequences with restricted affine gap penalties.

MOTIVATION: Given a genomic DNA sequence, it is still an open problem to determine its coding regions, i.e. the region consisting of exons and introns. The comparison of cDNA and genomic DNA helps the understanding of coding regions. For such an application, it might be adequate to use the restricted affine gap penalties which penalize long gaps with a constant penalty. RESULTS: Several techniques developed for solving the approximate string-matching problem are employed to yield efficient algorithms for computing the optimal alignment with restricted affine gap penalties. In particular, efficient algorithms can be derived based on the suffix automaton with failure transitions and on the diagonalwise monotonicity of the cost tables. We have implemented the above methods in C on Sun workstations running SunOS Unix. Preliminary experiments show that these approaches are very promising for aligning a cDNA sequence with a genomic DNA sequence. AVAILABILITY: Calign is available free of charge by anonymous ftp at: iubio.bio. indiana.edu, directory: molbio/align, files: calign.driver.c calign. c. Another URL reference for the files is http://iubio.bio.indiana.edu/soft/molbio/align/+ ++calign.c.     

GeneContent: software for whole-genome phylogenetic analysis

SUMMARY: GeneContent is a software system to infer the genome phylogeny based on an additive genome distance that can be estimated from the extended gene content data, which contains the genome-wide information (absence of a gene family, presence as single copy or presence as duplicates) across multiple species. GeneContent can also be used to explore the genome-wide evolutionary pattern of gene loss and proliferation. AVAILABILITY: Distribution packages of GeneContent for both Microsoft Windows and Linux operating systems are available at http://xgu.zool.iastate.edu CONTACT: xgu@iastate.edu.     

Sim4cc: a cross-species spliced alignment program

Advances in sequencing technologies have accelerated the sequencing of new genomes, far outpacing the generation of gene and protein resources needed to annotate them. Direct comparison and alignment of existing cDNA sequences from a related species is an effective and readily available means to determine genes in the new genomes. Current spliced alignment programs are inadequate for comparing sequences between different species, owing to their low sensitivity and splice junction accuracy. A new spliced alignment tool, sim4cc, overcomes problems in the earlier tools by incorporating three new features: universal spaced seeds, to increase sensitivity and allow comparisons between species at various evolutionary distances, and powerful splice signal models and evolutionarily-aware alignment techniques, to improve the accuracy of gene models. When tested on vertebrate comparisons at diverse evolutionary distances, sim4cc had significantly higher sensitivity compared to existing alignment programs, more than 10% higher than the closest competitor for some comparisons, while being comparable in speed to its predecessor, sim4. Sim4cc can be used in one-to-one or one-to-many comparisons of genomic and cDNA sequences, and can also be effectively incorporated into a high-throughput annotation engine, as demonstrated by the mapping of 64 000 Fagus grandifolia 454 ESTs and unigenes to the poplar genome.     

A Method for Construction, Cloning and Expression of Intron-Less Gene from Unannotated Genomic DNA

The present century has witnessed an unprecedented rise in genome sequences owing to various genome-sequencing programs. However, the same has not been replicated with cDNA or expressed sequence tags (ESTs). Hence, prediction of protein coding sequence of genes from this enormous collection of genomic sequences presents a significant challenge. While robust high throughput methods of cloning and expression could be used to meet protein requirements, lack of intron information creates a bottleneck. Computational programs designed for recognizing intron–exon boundaries for a particular organism or group of organisms have their own limitations. Keeping this in view, we describe here a method for construction of intron-less gene from genomic DNA in the absence of cDNA/EST information and organism-specific gene prediction program. The method outlined is a sequential application of bioinformatics to predict correct intron–exon boundaries and splicing by overlap extension PCR for spliced gene synthesis. The gene construct so obtained can then be cloned for protein expression. The method is simple and can be used for any eukaryotic gene expression.     

Homology-based gene structure prediction: simplified matching algorithm using a translated codon (tron) and improved accuracy by allowing for long gaps

MOTIVATION: Locating protein-coding exons (CDSs) on a eukaryotic genomic DNA sequence is the initial and an essential step in predicting the functions of the genes embedded in that part of the genome. Accurate prediction of CDSs may be achieved by directly matching the DNA sequence with a known protein sequence or profile of a homologous family member(s). RESULTS: A new convention for encoding a DNA sequence into a series of 23 possible letters (translated codon or tron code) was devised to improve this type of analysis. Using this convention, a dynamic programming algorithm was developed to align a DNA sequence and a protein sequence or profile so that the spliced and translated sequence optimally matches the reference the same as the standard protein sequence alignment allowing for long gaps. The objective function also takes account of frameshift errors, coding potentials, and translational initiation, termination and splicing signals. This method was tested on Caenorhabditis elegans genes of known structures. The accuracy of prediction measured in terms of a correlation coefficient (CC) was about 95% at the nucleotide level for the 288 genes tested, and 97. 0% for the 170 genes whose product and closest homologue share more than 30% identical amino acids. We also propose a strategy to improve the accuracy of prediction for a set of paralogous genes by means of iterative gene prediction and reconstruction of the reference profile derived from the predicted sequences. AVAILABILITY: The source codes for the program 'aln' written in ANSI-C and the test data will be available via anonymous FTP at ftp.genome.ad.jp/pub/genomenet/saitama-cc. CONTACT: gotoh@cancer-c.pref.saitama.jp     

Progress in maize gene discovery: a project update

The Maize Gene Discovery Project (MGDP) is a 5-year NSF-funded plant genome initiative that began in 1998. The MGDP collaboration involves researchers at six universities from diverse disciplines with the common goal of discovering new maize genes and developing tools for the phenotypic characterization of maize mutants. The project utilizes several approaches: EST sequencing, cDNA microarray production, and the discovery of gene function and genomic sequence through the use of a recombinant Mu1 transposon (RescueMu). Current achievements of the MGDP (NSF 98–72657) include the sequencing of over 120,000 maize ESTs from diverse cDNA libraries, and over 70,000 RescueMu flanking sequences, as well as the cataloguing of mutant seed and cob phenotypes of 23,000 maize ears, 6,200 families of maize seedlings, and 4,000 families of adult maize plants carrying MuDR/Mu and RescueMu insertion alleles. A consolidation of over 24,000 unique sequences from 19 libraries has been made into the first two of the planned set of four "Unigene" microarray slides. In addition, slides for four EST libraries have been produced. These microarray slides, EST clones, library plates of immortalized RescueMu bacterial cultures, and seed are all available online (http://www.zmdb.iastate.edu). The ZmDB website posts periodic assemblies of all maize EST and genomic sequences available from GenBank. ZmDB is also a portal for sequence analysis software designed to aid in gene discovery: MuSeqBox, GeneSeqer, and SplicePredictor . In addition, ZmDB contains links to other plant and genetics websites. Electronic Publication     

A generalized global alignment algorithm

MOTIVATION: Homologous sequences are sometimes similar over some regions but different over other regions. Homologous sequences have a much lower global similarity if the different regions are much longer than the similar regions. RESULTS: We present a generalized global alignment algorithm for comparing sequences with intermittent similarities, an ordered list of similar regions separated by different regions. A generalized global alignment model is defined to handle sequences with intermittent similarities. A dynamic programming algorithm is designed to compute an optimal general alignment in time proportional to the product of sequence lengths and in space proportional to the sum of sequence lengths. The algorithm is implemented as a computer program named GAP3 (Global Alignment Program Version 3). The generalized global alignment model is validated by experimental results produced with GAP3 on both DNA and protein sequences. The GAP3 program extends the ability of standard global alignment programs to recognize homologous sequences of lower similarity. AVAILABILITY: The GAP3 program is freely available for academic use at http://bioinformatics.iastate.edu/aat/align/align.html.     

A novel gene, DSCR5, from the distal Down syndrome critical region on chromosome 21q22.2.

Based on a detailed sequence of the distal Down syndrome critical region (DSCR), we predicted and molecularly cloned a novel gene, designated DSCR5. We determined the sequences of expressed sequence tags (ESTs) that almost matched the predicted cDNA sequence of DSCR5. Northern blot analysis showed that DSCR5 is expressed in several tissues including the liver, skeletal muscle, heart, pancreas and testis. To determine the 5'-end of DSCR5, the oligo-capping method was employed. Combining the EST sequence data and that from the oligo-capping experiments, we obtained the full-length cDNA sequence of DSCR5. DSCR5 had at least four types of alternatively spliced variants. According to the number of exons, they could be classified into two subtypes: DSCR5alpha and DSCR5beta. DSCR5alpha includes three splice variant subtypes, DSCR5alpha1, alpha2 and alpha3, which each has different first non-coding exon. In addition, the most abundantly isolated form, DSCR5alpha1, shows microheterogeneity of the mRNA start site. Comparison of the sequences between the predicted cDNA and the molecularly cloned cDNA revealed that the computer programs had limited validity to correctly predict the terminal exons. Thus, molecular cloning should always be required to complement the inadequacy of the computer predictions.     

Gene2EST: a BLAST2 server for searching expressed sequence tag (EST) databases with eukaryotic gene-sized queries

Expressed sequence tags (ESTs) are randomly sequenced cDNA clones. Currently, nearly 3 million human and 2 million mouse ESTs provide valuable resources that enable researchers to investigate the products of gene expression. The EST databases have proven to be useful tools for detecting homologous genes, for exon mapping, revealing differential splicing, etc. With the increasing availability of large amounts of poorly characterised eukaryotic (notably human) genomic sequence, ESTs have now become a vital tool for gene identification, sometimes yielding the only unambiguous evidence for the existence of a gene expression product. However, BLAST-based Web servers available to the general user have not kept pace with these developments and do not provide appropriate tools for querying EST databases with large highly spliced genes, often spanning 50 000-100 000 bases or more. Here we describe Gene2EST (http://woody.embl-heidelberg.de/gene2est/), a server that brings together a set of tools enabling efficient retrieval of ESTs matching large DNA queries and their subsequent analysis. RepeatMasker is used to mask dispersed repetitive sequences (such as Alu elements) in the query, BLAST2 for searching EST databases and Artemis for graphical display of the findings. Gene2EST combines these components into a Web resource targeted at the researcher who wishes to study one or a few genes to a high level of detail.     

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP-Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.