AGMIAL: implementing an annotation strategy for prokaryote genomes as a distributed system

We have implemented a genome annotation system for prokaryotes called AGMIAL. Our approach embodies a number of key principles. First, expert manual annotators are seen as a critical component of the overall system; user interfaces were cyclically refined to satisfy their needs. Second, the overall process should be orchestrated in terms of a global annotation strategy; this facilitates coordination between a team of annotators and automatic data analysis. Third, the annotation strategy should allow progressive and incremental annotation from a time when only a few draft contigs are available, to when a final finished assembly is produced. The overall architecture employed is modular and extensible, being based on the W3 standard Web services framework. Specialized modules interact with two independent core modules that are used to annotate, respectively, genomic and protein sequences. AGMIAL is currently being used by several INRA laboratories to analyze genomes of bacteria relevant to the food-processing industry, and is distributed under an open source license

CORG: a database for COmparative Regulatory Genomics

Sequence conservation in non-coding, upstream regions of orthologous genes from man and mouse is likely to reflect common regulatory DNA sites. Motivated by this assumption we have delineated a catalogue of conserved non-coding sequence blocks and provide the CORG-'COmparative Regulatory Genomics'-database. The data were computed based on statistically significant local suboptimal alignments of 15 kb regions upstream of the translation start sites of, currently, 10 793 pairs of orthologous genes. The resulting conserved non-coding blocks were annotated with EST matches for easier detection of non-coding mRNA and with hits to known transcription factor binding sites. CORG data are accessible from the ENSEMBL web site via a DAS service as well as a specially developed web service (http://corg.molgen.mpg.de) for query and interactive visualization of the conserved blocks and their annotation

Predicting Combinatorial Binding of Transcription Factors to Regulatory Elements in the Human Genome by Association Rule Mining

Cis-acting transcriptional regulatory elements in mammalian genomes typically contain specific combinations of binding sites for various transcription factors. Although some cisregulatory elements have been well studied, the combinations of transcription factors that regulate normal expression levels for the vast majority of the 20,000 genes in the human genome are unknown. We hypothesized that it should be possible to discover transcription factor combinations that regulate gene expression in concert by identifying over-represented combinations of sequence motifs that occur together in the genome. In order to detect combinations of transcription factor binding motifs, we developed a data mining approach based on the use of association rules, which are typically used in market basket analysis. We scored each segment of the genome for the presence or absence of each of 83 transcription factor binding motifs, then used association rule mining algorithms to mine this dataset, thus identifying frequently occurring pairs of distinct motifs within a segment. Results: Support for most pairs of transcription factor binding motifs was highly correlated across different chromosomes although pair significance varied. Known true positive motif pairs showed higher association rule support, confidence, and significance than background. Our subsets of high-confidence, high-significance mined pairs of transcription factors showed enrichment for co-citation in PubMed abstracts relative to all pairs, and the predicted associations were often readily verifiable in the literature. Conclusion: Functional elements in the genome where transcription factors bind to regulate expression in a combinatorial manner are more likely to be predicted by identifying statistically and biologically significant combinations of transcription factor binding motifs than by simply scanning the genome for the occurrence of binding sites for a single transcription factor.NIAAA Alcohol Training GrantNational Science FoundationCellular and Molecular Biolog

Defining functional DNA elements in the human genome

With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease

The origins, evolution, and functional potential of alternative splicing in vertebrates.

Alternative splicing (AS) has the potential to greatly expand the functional repertoire of mammalian transcriptomes. However, few variant transcripts have been characterized functionally, making it difficult to assess the contribution of AS to the generation of phenotypic complexity and to study the evolution of splicing patterns. We have compared the AS of 309 protein-coding genes in the human ENCODE pilot regions against their mouse orthologs in unprecedented detail, utilizing traditional transcriptomic and RNAseq data. The conservation status of every transcript has been investigated, and each functionally categorized as coding (separated into coding sequence [CDS] or nonsense-mediated decay [NMD] linked) or noncoding. In total, 36.7% of human and 19.3% of mouse coding transcripts are species specific, and we observe a 3.6 times excess of human NMD transcripts compared with mouse; in contrast to previous studies, the majority of species-specific AS is unlinked to transposable elements. We observe one conserved CDS variant and one conserved NMD variant per 2.3 and 11.4 genes, respectively. Subsequently, we identify and characterize equivalent AS patterns for 22.9% of these CDS or NMD-linked events in nonmammalian vertebrate genomes, and our data indicate that functional NMD-linked AS is more widespread and ancient than previously thought. Furthermore, although we observe an association between conserved AS and elevated sequence conservation, as previously reported, we emphasize that 30% of conserved AS exons display sequence conservation below the average score for constitutive exons. In conclusion, we demonstrate the value of detailed comparative annotation in generating a comprehensive set of AS transcripts, increasing our understanding of AS evolution in vertebrates. Our data supports a model whereby the acquisition of functional AS has occurred throughout vertebrate evolution and is considered alongside amino acid change as a key mechanism in gene evolution

A purely bioinformatic pipeline for the prediction of mammalian odorant receptor gene enhancers

Background In most mammals, a vast array of genes coding for chemosensory receptors mediates olfaction. Odorant receptor (OR) genes generally constitute the largest multifamily (> 1100 intact members in the mouse). From the whole pool, each olfactory neuron expresses a single OR allele following poorly characterized mechanisms termed OR gene choice. OR genes are found in genomic aggregations known as clusters. Nearby enhancers, named elements, are crucial regulators of OR gene choice. Despite their importance, searching for new elements is burdensome. Other chemosensory receptor genes responsible for smell adhere to expression modalities resembling OR gene choice, and are arranged in genomic clusters - often with chromosomal linkage to OR genes. Still, no elements are known for them. Results Here we present an inexpensive framework aimed at predicting elements. We redefine cluster identity by focusing on multiple receptor gene families at once, and exemplify thirty - not necessarily OR-exclusive - novel candidate enhancers. Conclusions The pipeline we introduce could guide future in vivo work aimed at discovering/validating new elements. In addition, our study provides an updated and comprehensive classification of all genomic loci responsible for the transduction of olfactory signals in mammals

GenomeTrafac: a whole genome resource for the detection of transcription factor binding site clusters associated with conventional and microRNA encoding genes conserved between mouse and human gene orthologs

Transcriptional cis-regulatory control regions frequently are found within non-coding DNA segments conserved across multi-species gene orthologs. Adopting a systematic gene-centric pipeline approach, we report here the development of a web-accessible database resource—GenomeTraFac ()—that allows genome-wide detection and characterization of compositionally similar cis-clusters that occur in gene orthologs between any two genomes for both microRNA genes as well as conventional RNA-encoding genes. Each ortholog gene pair can be scanned to visualize overall conserved sequence regions, and within these, the relative density of conserved cis-element motif clusters form graph peak structures. The results of these analyses can be mined en masse to identify most frequently represented cis-motifs in a list of genes. The system also provides a method for rapid evaluation and visualization of gene model-consistency between orthologs, and facilitates consideration of the potential impact of sequence variation in conserved non-coding regions to impact complex cis-element structures. Using the mouse and human genomes via the NCBI Reference Sequence database and the Sanger Institute miRBase, the system demonstrated the ability to identify validated transcription factor targets within promoter and distal genomic regulatory regions of both conventional and microRNA genes

Hunter-Gatherer-Annotator science : characterizing regulatory elements in the genome of dog and zebrafish with public and not yet public data

In order to study gene regulation, large amounts of sequencing data are necessary. We can either generate (hunt) them ourselves or use (gather) publicly available data sets. In order to guarantee the reliability and reusability of the hunted and gathered data, we need to also annotate them with the correct metadata. In this thesis, I will touch on all three of these aspects. I was part of two international consortia which applied these approaches to two different model organisms. The DANIO-CODE consortium was initiated to systematically annotate the zebrafish genome. Similarly, the Dog Genome Annotation (DoGA) project aims to improve the annotation of genomic elements in the dog genome. Both zebrafish and dogs are popular model organisms for studying biological processes and pathologies in humans. Despite their popularity, both organisms lack a large-scale annotation of regulatory elements. Before analyzing any data, we designed an annotation structure that captures all aspects of a sequencing experiment that are essential for the processing and analysis of the data. We implemented this structure in a web-platform, which allows easy upload, query, and download of the sequencing data and associated metadata. We present the structure and implementation in Study I, which also contains a comparison to similar and well-established annotation schemata. We use this annotation structure and the web platform for Study II to collect sequencing data from 1,803 samples from 38 different research groups looking from transcriptomic, epigenomic, and methylomic perspectives at different stages of zebrafish development. We identified more than 140,000 new cis-regulatory elements active during development and provide them together with the sequencing data and genome browser tracks as a resource for the community. In Study III, we present a biobank for dog tissues established for the DoGA consortium. For both Study III and Study IV, we used 88 and 37 tissues from the biobank, respectively, to catalog promoter regions and their tissue activity using STRT and CAGE-seq. In Study III we also present the web-platform, based on the structure in Study I, where we make the data and the corresponding metadata available. In Study IV, we used the data from CAGE-seq to also identify active enhancer regions and their corresponding tissue activity. We identify regulatory networks between enhancers and promoters and show their conservation in human

Comparative promoter region analysis powered by CORG

BACKGROUND: Promoters are key players in gene regulation. They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG , a framework for studying upstream regions including untranslated exons (5' UTR). DESCRIPTION: The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences. As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server . The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set. CONCLUSION: The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites)