Comparative Genomic Information Research Articles

Prediction of novel microRNA genes in cancer-associated genomic regions—a combined computational and experimental approach

The majority of existing computational tools rely on sequence homology and/or structural similarity to identify novel microRNA (miRNA) genes. Recently supervised algorithms are utilized to address this problem, taking into account sequence, structure and comparative genomics information. In most of these studies miRNA gene predictions are rarely supported by experimental evidence and prediction accuracy remains uncertain. In this work we present a new computational tool (SSCprofiler) utilizing a probabilistic method based on Profile Hidden Markov Models to predict novel miRNA precursors. Via the simultaneous integration of biological features such as sequence, structure and conservation, SSCprofiler achieves a performance accuracy of 88.95% sensitivity and 84.16% specificity on a large set of human miRNA genes. The trained classifier is used to identify novel miRNA gene candidates located within cancer-associated genomic regions and rank the resulting predictions using expression information from a full genome tiling array. Finally, four of the top scoring predictions are verified experimentally using northern blot analysis. Our work combines both analytical and experimental techniques to show that SSCprofiler is a highly accurate tool which can be used to identify novel miRNA gene candidates in the human genome. SSCprofiler is freely available as a web service at http://www.imbb.forth.gr/SSCprofiler.html.

Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure

BackgroundTetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.ResultsWe addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.ConclusionWe report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.

Identification of multiple independent horizontal gene transfers into poxviruses using a comparative genomics approach

BackgroundPoxviruses are important pathogens of humans, livestock and wild animals. These large dsDNA viruses have a set of core orthologs whose gene order is extremely well conserved throughout poxvirus genera. They also contain many genes with sequence and functional similarity to host genes which were probably acquired by horizontal gene transfer.Although phylogenetic trees can indicate the occurrence of horizontal gene transfer and even uncover multiple events, their use may be hampered by uncertainties in both the topology and the rooting of the tree. We propose to use synteny conservation around the horizontally transferred gene (HTgene) to distinguish between single and multiple events.ResultsHere we devise a method that incorporates comparative genomic information into the investigation of horizontal gene transfer, and we apply this method to poxvirus genomes. We examined the synteny conservation around twenty four pox genes that we identified, or which were reported in the literature, as candidate HTgenes. We found support for multiple independent transfers into poxviruses for five HTgenes. Three of these genes are known to be important for the survival of the virus in or out of the host cell and one of them increases susceptibility to some antiviral drugs.ConclusionIn related genomes conserved synteny information can provide convincing evidence for multiple independent horizontal gene transfer events even in the absence of a robust phylogenetic tree for the HTgene.

CTCFBSDB: a CTCF-binding site database for characterization of vertebrate genomic insulators

Recent studies on transcriptional control of gene expression have pinpointed the importance of long-range interactions and three-dimensional organization of chromatins within the nucleus. Distal regulatory elements such as enhancers may activate transcription over long distances; hence, their action must be restricted within appropriate boundaries to prevent illegitimate activation of non-target genes. Insulators are DNA elements with enhancer-blocking and/or chromatin-bordering functions. In vertebrates, the versatile transcription regulator CCCTC-binding factor (CTCF) is the only identified trans-acting factor that confers enhancer-blocking insulator activity. CTCF-binding sites were found to be commonly distributed along the vertebrate genomes. We have constructed a CTCF-binding site database (CTCFBSDB) to characterize experimentally identified and computationally predicted CTCF-binding sties. Biological knowledge and data from multiple resources have been integrated into the database, including sequence data, genetic polymorphisms, function annotations, histone methylation profiles, gene expression profiles and comparative genomic information. A web-based user interface was implemented for data retrieval, analysis and visualization. In silico prediction of CTCF-binding motifs is provided to facilitate the identification of candidate insulators in the query sequences submitted by users. The database can be accessed at http://insulatordb.utmem.edu/

The Princeton Protein Orthology Database (P-POD): A Comparative Genomics Analysis Tool for Biologists

Many biological databases that provide comparative genomics information and tools are now available on the internet. While certainly quite useful, to our knowledge none of the existing databases combine results from multiple comparative genomics methods with manually curated information from the literature. Here we describe the Princeton Protein Orthology Database (P-POD, http://ortholog.princeton.edu), a user-friendly database system that allows users to find and visualize the phylogenetic relationships among predicted orthologs (based on the OrthoMCL method) to a query gene from any of eight eukaryotic organisms, and to see the orthologs in a wider evolutionary context (based on the Jaccard clustering method). In addition to the phylogenetic information, the database contains experimental results manually collected from the literature that can be compared to the computational analyses, as well as links to relevant human disease and gene information via the OMIM, model organism, and sequence databases. Our aim is for the P-POD resource to be extremely useful to typical experimental biologists wanting to learn more about the evolutionary context of their favorite genes. P-POD is based on the commonly used Generic Model Organism Database (GMOD) schema and can be downloaded in its entirety for installation on one's own system. Thus, bioinformaticians and software developers may also find P-POD useful because they can use the P-POD database infrastructure when developing their own comparative genomics resources and database tools.

The ASAP II database: analysis and comparative genomics of alternative splicing in 15 animal species

We have greatly expanded the Alternative Splicing Annotation Project (ASAP) database: (i) its human alternative splicing data are expanded ∼3-fold over the previous ASAP database, to nearly 90 000 distinct alternative splicing events; (ii) it now provides genome-wide alternative splicing analyses for 15 vertebrate, insect and other animal species; (iii) it provides comprehensive comparative genomics information for comparing alternative splicing and splice site conservation across 17 aligned genomes, based on UCSC multigenome alignments; (iv) it provides an ∼2- to 3-fold expansion in detection of tissue-specific alternative splicing events, and of cancer versus normal specific alternative splicing events. We have also constructed a novel database linking orthologous exons and orthologous introns between genomes, based on multigenome alignment of 17 animal species. It can be a valuable resource for studies of gene structure evolution. ASAP II provides a new web interface enabling more detailed exploration of the data, and integrating comparative genomics information with alternative splicing data. We provide a set of tools for advanced data-mining of ASAP II with Pygr (the Python Graph Database Framework for Bioinformatics) including powerful features such as graph query, multigenome alignment query, etc. ASAP II is available at .

Classification of real and pseudo microRNA precursors using local structure-sequence features and support vector machine

BackgroundMicroRNAs (miRNAs) are a group of short (~22 nt) non-coding RNAs that play important regulatory roles. MiRNA precursors (pre-miRNAs) are characterized by their hairpin structures. However, a large amount of similar hairpins can be folded in many genomes. Almost all current methods for computational prediction of miRNAs use comparative genomic approaches to identify putative pre-miRNAs from candidate hairpins. Ab initio method for distinguishing pre-miRNAs from sequence segments with pre-miRNA-like hairpin structures is lacking. Being able to classify real vs. pseudo pre-miRNAs is important both for understanding of the nature of miRNAs and for developing ab initio prediction methods that can discovery new miRNAs without known homology.ResultsA set of novel features of local contiguous structure-sequence information is proposed for distinguishing the hairpins of real pre-miRNAs and pseudo pre-miRNAs. Support vector machine (SVM) is applied on these features to classify real vs. pseudo pre-miRNAs, achieving about 90% accuracy on human data. Remarkably, the SVM classifier built on human data can correctly identify up to 90% of the pre-miRNAs from other species, including plants and virus, without utilizing any comparative genomics information.ConclusionThe local structure-sequence features reflect discriminative and conserved characteristics of miRNAs, and the successful ab initio classification of real and pseudo pre-miRNAs opens a new approach for discovering new miRNAs.

Facilitating genome navigation: survey sequencing and dense radiation-hybrid gene mapping

Accurate and comprehensive sequence coverage for large genomes has been restricted to only a few species of specific interest. Lower sequence coverage (survey sequencing) of related species can yield a wealth of information about gene content and putative regulatory elements. But survey sequences lack long-range continuity and provide only a fragmented view of a genome. Here we show the usefulness of combining survey sequencing with dense radiation-hybrid (RH) maps for extracting maximum comparative genome information from model organisms. Based on results from the canine system, we propose that from now on all low-pass sequencing projects should be accompanied by a dense, gene-based RH map-construction effort to extract maximum information from the genome with a marginal extra cost.

Zea mays ontology – a database of international terms

Data retrieval, comprehension and sharing within and between plant-based databases are essential to exploit comparative genomic information to elucidate functional aspects of plant biology and to conduct studies of synteny and homology. However, the functionality is often hampered by the variability of terms used to describe comparable objects. The Zea mays Plant Structure Ontology database is designed to overcome this problem via the provision of a controlled vocabulary that facilitates knowledge sharing. It comprises international botanical terms, references, synonyms, and phylogenetic information and is open-source.

The Mouse Genome Database (MGD): the model organism database for the laboratory mouse.

The Mouse Genome Database (MGD) is the community database resource for the laboratory mouse, a key model organism for interpreting the human genome and for understanding human biology and disease (http://www.informatics.jax.org). MGD strives to provide a highly curated, highly integrated information resource that not only includes the consensus view of current knowledge about the mouse, but also provides comparative genomic information particularly for human and rat genomes. MGD includes extensive information about mouse genes, supporting all gene attribute assertions with experimental data, statements of evidence and citation. Detailed information about alleles and mouse mutants includes genotype, molecular variant and phenotype descriptions. Extensive collaboration with other data providers such as NCBI, RIKEN and SWISS-PROT provides standardization of gene:sequence associations and robust interconnections between large information systems based on shared sequence curation. Recent integration of large datasets of mouse full-length cDNAs and radiation-hybrid mapped ESTs, the continued development and use of extensive structured vocabularies and the expansion of the representation of phenotypes highlight this year's developments.

A receptor tyrosine kinase from choanoflagellates: molecular insights into early animal evolution.

The evolution of the Metazoa from protozoans is one of the major milestones in life's history. The genetic and developmental events involved in this evolutionary transition are unknown but may have involved the evolution of genes required for signaling and gene regulation in metazoans. The genome of animal ancestors may be reconstructed by identification of animal genes that are shared with related eukaryotes, particularly those that share a more recent ancestry and cell biology with animals. The choanoflagellates have long been suspected to be closer relatives of animals than are fungi, the closest outgroup of animals for which comparative genomic information is available. Phylogenetic analyses of choanoflagellate and animal relationships based on small subunit rDNA sequence, however, have yielded ambiguous and conflicting results. We find that analyses of four conserved proteins from a unicellular choanoflagellate, Monosiga brevicollis, provide robust support for a close relationship between choanoflagellates and Metazoa, suggesting that comparison of the complement of expressed genes from choanoflagellates and animals may be informative concerning the early evolution of metazoan genomes. We have discovered in M. brevicollis the first receptor tyrosine kinase (RTK), to our knowledge, identified outside of the Metazoa, MBRTK1. The architecture of MBRTK1, which includes multiple extracellular ligand-binding domains, resembles that of RTKs in sponges and humans and suggests the ability to receive and transduce signals. Thus, choanoflagellates express genes involved in animal development that are not found in other eukaryotes and that may be linked to the origin of the Metazoa.

Genomic tools to improve parasite resistance

The natural genetic variability of the ruminant immune system provides a feasible means to control gastrointestinal (GI) parasite infection without anthelmintics. However, the paradigm of traditional selection has not been effectively applied to the moderately heritable traits of parasite resistance ( h≈0.3) due to the difficulty and expense of gathering accurate phenotypes in a commercial production setting. These characteristics make host traits related to GI nematode infection ideal candidates for genomics-based research. To initiate explanation of important allelic differences, economic trait loci (ETL) are being identified and mapped using a resource population of Angus cattle segregating for GI nematode resistance and susceptibility to the two most common nematode parasites of US cattle, Ostertagia ostertagi and Cooperia oncophora. The population is composed of five generations of half-sib progeny with complete phenotypic records produced from controlled infections. To detect the genomic locations of the three distinct phenotypic traits being expressed (innately immune, acquired immune, and immunologically non-responsive), genotypes have been generated for DNA markers ( N=199) spaced at regular intervals (∼20 cm intervals) throughout the entire genome (3000 cm). Although initial ETL detection may be limited by half-sib family size, the unique structure of this population provides additional statistical power for refining map position of potential ETL. After allele frequency and contribution to phenotype are determined in this population, marker tests associated with ETL most beneficial for controlling parasite infection can be accurately used for selection. Comparative map and functional genomic information from humans and other species of biomedical importance will be utilized in further investigations to elucidate the genes underlying ETL.

Comparative Analysis on the Conservation of Two Orthologous Genome Regions

Recently, draft sequences of human and mouse genome have been released. Such genomic sequence data are increasing very rapidly and effective annotation methods are required. Investigating orthologous contigs of two genomes can be very useful to understand their functional similarities and differences. Especially, as the mouse becomes a leading model for studying disease processes in humans, the emerging mouse genome data have become valuable resources for functional studies [1]. So far there have been several approaches to analyze important CDS (coding sequence) regions and non-CDS (e.g. promoter, polyA, etc.) regions individually. Here, we tried to systemically analyze coding and non-coding human and mouse orthologous regions with putative annotations by several prediction tools (e.g. Genscan, RepeatMasker, Sim4, etc.) accompanying database search (TIGR Gene Indices [7]) as well as with known annotations extracted from Genbank flat data files. Finally web-based visualization tools were applied to display comparative genomic information effectively.

The conserved genome organisation of non-falciparum malaria species: the need to know more

The current knowledge on genomes of non- falciparum malaria species and the potential of model malaria parasites for functional analyses are reviewed and compared with those of the most pathogenic human parasite, Plasmodium falciparum. There are remarkable similarities in overall genome composition among the different species at the level of chromosome organisation and chromosome number, conserved order of individual genes, and even conserved functions of specific gene domains and regulatory control elements. With the initiative taken to sequence the genome of P. falciparum, a wealth of information is already becoming available to the scientific community. In order to exploit the biological information content of a complete genome sequence, simple storage of the bulk of sequence data will be inadequate. The requirement for functional analyses to determine the biological role of the open reading frames is commonly accepted and knowledge of the genomes of the animal model malaria species will facilitate these analyses. Detailed comparative genome information and sequencing of additional Plasmodium genomes will provide a deeper insight into the evolutionary history of the species, the biology of the parasite, and its interactions with the mammalian host and mosquito vector. Therefore, an extended and integrated approach will enhance our knowledge of malaria and will ultimately lead to a more rational approach that identifies and evaluates new targets for anti-malarial drug and vaccine development.