Summary

At least half of the human genome is derived from repetitive elements, which are often lineage specific and silenced by a variety of genetic and epigenetic mechanisms. Using a transchromosomic mouse strain that transmits an almost complete single copy of human chromosome 21 via the female germline, we show that a heterologous regulatory environment can transcriptionally activate transposon-derived human regulatory regions. In the mouse nucleus, hundreds of locations on human chromosome 21 newly associate with activating histone modifications in both somatic and germline tissues, and influence the gene expression of nearby transcripts. These regions are enriched with primate and human lineage-specific transposable elements, and their activation corresponds to changes in DNA methylation at CpG dinucleotides. This study reveals the latent regulatory potential of the repetitive human genome and illustrates the species specificity of mechanisms that control it.

Highlights

► A mouse carrying human chromosome 21 fails to repress primate-specific repeats ► The lack of repression was revealed by H3K4me3 and transcription factor binding ► Activation corresponded to a decrease in CpG methylation ► Primate-specific repeats activated in human testes were activated in the Tc1 mouse

The Ensembl project (http://www.ensembl.org) provides genome information for sequenced chordate genomes with a particular focus on human, mouse, zebrafish and rat. Our resources include evidenced-based gene sets for all supported species; large-scale whole genome multiple species alignments across vertebrates and clade-specific alignments for eutherian mammals, primates, birds and fish; variation data resources for 17 species and regulation annotations based on ENCODE and other data sets. Ensembl data are accessible through the genome browser at http://www.ensembl.org and through other tools and programmatic interfaces.

Much has changed in the last two years at DGVa (http://www.ebi.ac.uk/dgva) and dbVar (http://www.ncbi.nlm.nih.gov/dbvar). We are now processing direct submissions rather than only curating data from the literature and our joint study catalog includes data from over 100 studies in 11 organisms. Studies from human dominate with data from control and case populations, tumor samples as well as three large curated studies derived from multiple sources. During the processing of these data, we have made improvements to our data model, submission process and data representation. Additionally, we have made significant improvements in providing access to these data via web and FTP interfaces.

The 1000 Genomes Project was launched as one of the largest distributed data collection and analysis projects ever undertaken in biology. In addition to the primary scientific goals of creating both a deep catalogue of human genetic variation and extensive methods to accurately discover and characterize variation using new sequencing technologies, the project makes all of its data publicly available for community use. The project data coordination center has developed and deployed several tools to enable widespread data access.

Detecting genetic variants that are highly divergent from a reference sequence remains a major challenge in genome sequencing. We introduce de novo assembly algorithms using colored de Bruijn graphs for detecting and genotyping simple and complex genetic variants in an individual or population. We provide an efficient software implementation, Cortex; the first de novo assembler capable of assembling multiple eukaryote genomes simultaneously. Four applications of Cortex are presented. First, we detect and validate both simple and complex structural variation in a high coverage human genome. Second, we identify over 3Mb of novel sequence in pooled low-coverage population sequence data from the 1000 Genomes Project. Third, we show how population information from 10 chimpanzees enables accurate variant calls without a reference sequence. Finally, we estimate classical HLA genotypes at HLA-B, the most variable gene in the human genome.

Comparison of related genomes has emerged as a powerful lens for genome interpretation. Here, we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and report constrained elements covering ~4.2% of the genome. We use evolutionary signatures and comparison with experimental datasets to suggest candidate functions for ~60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events, and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements, and ~1,000 primate- and human-accelerated elements. Overlap with disease-associated variants suggests our findings will be relevant for studies of human biology and health.

A response to 2x genomes - depth does matter by MC Milinkovitch, R Helaers, E Depiereux, AC Tzika and T Gabaldón. Genome Biol 2010, 11:R16.

Summary

CTCF-binding locations represent regulatory sequences that are highly constrained over the course of evolution. To gain insight into how these DNA elements are conserved and spread through the genome, we defined the full spectrum of CTCF-binding sites, including a 33/34-mer motif, and identified over five thousand highly conserved, robust, and tissue-independent CTCF-binding locations by comparing ChIP-seq data from six mammals. Our data indicate that activation of retroelements has produced species-specific expansions of CTCF binding in rodents, dogs, and opossum, which often functionally serve as chromatin and transcriptional insulators. We discovered fossilized repeat elements flanking deeply conserved CTCF-binding regions, indicating that similar retrotransposon expansions occurred hundreds of millions of years ago. Repeat-driven dispersal of CTCF binding is a fundamental, ancient, and still highly active mechanism of genome evolution in mammalian lineages.

PaperClip

Graphical Abstract

Highlights

► CTCF-binding locations are highly conserved across mammals ► New locations for CTCF binding are carried by SINE repeats in many mammals ► Ancient and newly born CTCF-binding events similarly demarcate chromatin barriers ► Retroelements can reposition organizing elements throughout the genome

Evolutionary analysis of six divergent mammals uncovers both highly conserved and species-specific binding locations of the chromatin organizer, CTCF. This study reveals that activation of retroelements drives genome evolution by repositioning genome-organizing elements.

The Ensembl project (http://www.ensembl.org) provides genome resources for chordate genomes with a particular focus on human genome data as well as data for key model organisms such as mouse, rat and zebrafish. Five additional species were added in the last year including gibbon (Nomascus leucogenys) and Tasmanian devil (Sarcophilus harrisii) bringing the total number of supported species to 61 as of Ensembl release 64 (September 2011). Of these, 55 species appear on the main Ensembl website and six species are provided on the Ensembl preview site (Pre!Ensembl; http://pre.ensembl.org) with preliminary support. The past year has also seen improvements across the project.

Background

Rare coding variants constitute an important class of human genetic variation, but are underrepresented in current databases that are based on small population samples. Recent studies show that variants altering amino acid sequence and protein function are enriched at low variant allele frequency, 2 to 5%, but because of insufficient sample size it is not clear if the same trend holds for rare variants below 1% allele frequency.

Results

The 1000 Genomes Exon Pilot Project has collected deep-coverage exon-capture data in roughly 1,000 human genes, for nearly 700 samples. Although medical whole-exome projects are currently afoot, this is still the deepest reported sampling of a large number of human genes with next-generation technologies. According to the goals of the 1000 Genomes Project, we created effective informatics pipelines to process and analyze the data, and discovered 12,758 exonic SNPs, 70% of them novel, and 74% below 1% allele frequency in the seven population samples we examined. Our analysis confirms that coding variants below 1% allele frequency show increased population-specificity and are enriched for functional variants.

Conclusions

This study represents a large step toward detecting and interpreting low frequency coding variation, clearly lays out technical steps for effective analysis of DNA capture data, and articulates functional and population properties of this important class of genetic variation.

For a number of years the BioMart data warehousing system has proven to be a valuable resource for scientists seeking a fast and versatile means of accessing the growing volume of genomic data provided by the Ensembl project. The launch of the Ensembl Genomes project in 2009 complemented the Ensembl project by utilizing the same visualization, interactive and programming tools to provide users with a means for accessing genome data from a further five domains: protists, bacteria, metazoa, plants and fungi. The Ensembl and Ensembl Genomes BioMarts provide a point of access to the high-quality gene annotation, variation data, functional and regulatory annotation and evolutionary relationships from genomes spanning the taxonomic space. This article aims to give a comprehensive overview of the Ensembl and Ensembl Genomes BioMarts as well as some useful examples and a description of current data content and future objectives.

Database URLs: http://www.ensembl.org/biomart/martview/; http://metazoa.ensembl.org/biomart/martview/; http://plants.ensembl.org/biomart/martview/; http://protists.ensembl.org/biomart/martview/; http://fungi.ensembl.org/biomart/martview/; http://bacteria.ensembl.org/biomart/martview/

Conserved vertebrate transcription factors (TFs) direct gene expression by binding to DNA regulatory regions. To explore the evolution of gene regulation, we experimentally determined the genome-wide occupancy of two TFs, CEBPA and HNF4A, in livers of multiple vertebrates. Although each TF displays highly conserved DNA binding preferences, most binding is species-specific, and aligned binding events present in all five species are rare. Regions near genes with expression levels dependent on a TF are often bound by the TF in multiple species, yet show no enhanced DNA sequence constraint. Binding divergence between species can be largely explained by sequence changes to the the bound motifs. Among the binding events lost in one lineage, only half are recovered by another binding event within 10 kilobases. Our results reveal large interspecies differences in transcriptional regulation and provide insight into their evolution.

The Ensembl project (http://www.ensembl.org) seeks to enable genomic science by providing high quality, integrated annotation on chordate and selected eukaryotic genomes within a consistent and accessible infrastructure. All supported species include comprehensive, evidence-based gene annotations and a selected set of genomes includes additional data focused on variation, comparative, evolutionary, functional and regulatory annotation. The most advanced resources are provided for key species including human, mouse, rat and zebrafish reflecting the popularity and importance of these species in biomedical research. As of Ensembl release 59 (August 2010), 56 species are supported of which 5 have been added in the past year. Since our previous report, we have substantially improved the presentation and integration of both data of disease relevance and the regulatory state of different cell types.

The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome.

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.

Author Summary

In contrast to the compact sequence of viruses and bacteria, determining the complete genome sequence of complex vertebrate genomes can be a daunting task. With the advent of “next-generation” sequencing platforms, it is now possible to rapidly sequence and assemble a vertebrate genome, especially for species for which genomic resources—genetic maps and markers—are currently available. We used a combination of two next-generation sequencing platforms, Roche 454 and Illumina GAII, and unique assembly tools to sequence the genome of the agriculturally important turkey, Meleagris gallopavo. Our draft assembly comprises approximately 1.1 gigabases of which 917 megabytes are assigned to specific chromosomes. Comparisons of the turkey genome sequence with those of the chicken, Gallus gallus, and the zebra finch, Taeniopygia guttata, provide insights into the evolution of the avian lineage. This genome sequence will facilitate discovery of agriculturally important genetic variants.

Here we describe the Genome Variation Format (GVF) and the 10Gen dataset. GVF, an extension of Generic Feature Format version 3 (GFF3), is a simple tab-delimited format for DNA variant files, which uses Sequence Ontology to describe genome variation data. The 10Gen dataset, ten human genomes in GVF format, is freely available for community analysis from the Sequence Ontology website and from an Amazon elastic block storage (EBS) snapshot for use in Amazon's EC2 cloud computing environment.

CHD7 is one of nine members of the chromodomain helicase DNA–binding domain family of ATP–dependent chromatin remodeling enzymes found in mammalian cells. De novo mutation of CHD7 is a major cause of CHARGE syndrome, a genetic condition characterized by multiple congenital anomalies. To gain insights to the function of CHD7, we used the technique of chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP–Seq) to map CHD7 sites in mouse ES cells. We identified 10,483 sites on chromatin bound by CHD7 at high confidence. Most of the CHD7 sites show features of gene enhancer elements. Specifically, CHD7 sites are predominantly located distal to transcription start sites, contain high levels of H3K4 mono-methylation, found within open chromatin that is hypersensitive to DNase I digestion, and correlate with ES cell-specific gene expression. Moreover, CHD7 co-localizes with P300, a known enhancer-binding protein and strong predictor of enhancer activity. Correlations with 18 other factors mapped by ChIP–seq in mouse ES cells indicate that CHD7 also co-localizes with ES cell master regulators OCT4, SOX2, and NANOG. Correlations between CHD7 sites and global gene expression profiles obtained from Chd7+/+, Chd7+/−, and Chd7−/− ES cells indicate that CHD7 functions at enhancers as a transcriptional rheostat to modulate, or fine-tune the expression levels of ES–specific genes. CHD7 can modulate genes in either the positive or negative direction, although negative regulation appears to be the more direct effect of CHD7 binding. These data indicate that enhancer-binding proteins can limit gene expression and are not necessarily co-activators. Although ES cells are not likely to be affected in CHARGE syndrome, we propose that enhancer-mediated gene dysregulation contributes to disease pathogenesis and that the critical CHD7 target genes may be subject to positive or negative regulation.

Author Summary

The gene encoding chromodomain helicase DNA–binding protein 7 (CHD7) is required for normal mammalian development. In humans, genetic mutations in CHD7 lead to CHARGE syndrome, a disorder characterized by multiple birth defects. In previous studies, CHD7 was shown to localize to the cell nucleus and bind to specific sites on chromatin. However, the genome-wide distribution of CHD7 on chromatin and its function are not known. Here, we identified 10,483 sites on chromatin bound by CHD7 in mouse embryonic stem cells. Many of these sites are gene enhancer elements suspected to be involved in turning on genes. We show CHD7 functions at these loci to fine-tune the levels of genes that are specifically expressed in mouse ES cells. This modulation is mediated through several proteins that bind together with CHD7 at enhancer elements and can occur in either direction. These findings suggest CHARGE syndrome is the result of key genes that are improperly expressed during development. These key genes are currently unknown but are likely to be tissue-specific and may be upregulated or downregulated in response to CHD7 mutation.

The recent explosion of biological data and the concomitant proliferation of distributed databases make it challenging for biologists and bioinformaticians to discover the best data resources for their needs, and the most efficient way to access and use them. Despite a rapid acceleration in uptake of syntactic and semantic standards for interoperability, it is still difficult for users to find which databases support the standards and interfaces that they need. To solve these problems, several groups are developing registries of databases that capture key metadata describing the biological scope, utility, accessibility, ease-of-use and existence of web services allowing interoperability between resources. Here, we describe some of these initiatives including a novel formalism, the Database Description Framework, for describing database operations and functionality and encouraging good database practise. We expect such approaches will result in improved discovery, uptake and utilization of data resources.

Database URL: http://www.casimir.org.uk/casimir_ddf

Summary: A tool to predict the effect that newly discovered genomic variants have on known transcripts is indispensible in prioritizing and categorizing such variants. In Ensembl, a web-based tool (the SNP Effect Predictor) and API interface can now functionally annotate variants in all Ensembl and Ensembl Genomes supported species.

Availability: The Ensembl SNP Effect Predictor can be accessed via the Ensembl website at http://www.ensembl.org/. The Ensembl API (http://www.ensembl.org/info/docs/api/api_installation.html for installation instructions) is open source software.

Contact: wm2@ebi.ac.uk; fiona@ebi.ac.uk

Supplementary information: Supplementary data are available at Bioinformatics online.

Background

Advances in sequencing and genotyping technologies are leading to the widespread availability of multi-species variation data, dense genotype data and large-scale resequencing projects. The 1000 Genomes Project and similar efforts in other species are challenging the methods previously used for storage and manipulation of such data necessitating the redesign of existing genome-wide bioinformatics resources.

Results

Ensembl has created a database and software library to support data storage, analysis and access to the existing and emerging variation data from large mammalian and vertebrate genomes. These tools scale to thousands of individual genome sequences and are integrated into the Ensembl infrastructure for genome annotation and visualisation. The database and software system is easily expanded to integrate both public and non-public data sources in the context of an Ensembl software installation and is already being used outside of the Ensembl project in a number of database and application environments.

Conclusions

Ensembl's powerful, flexible and open source infrastructure for the management of variation, genotyping and resequencing data is freely available at http://www.ensembl.org.

Background

The Ensembl project produces updates to its comparative genomics resources with each of its several releases per year. During each release cycle approximately two weeks are allocated to generate all the genomic alignments and the protein homology predictions. The number of calculations required for this task grows approximately quadratically with the number of species. We currently support 50 species in Ensembl and we expect the number to continue to grow in the future.

Results

We present eHive, a new fault tolerant distributed processing system initially designed to support comparative genomic analysis, based on blackboard systems, network distributed autonomous agents, dataflow graphs and block-branch diagrams. In the eHive system a MySQL database serves as the central blackboard and the autonomous agent, a Perl script, queries the system and runs jobs as required. The system allows us to define dataflow and branching rules to suit all our production pipelines. We describe the implementation of three pipelines: (1) pairwise whole genome alignments, (2) multiple whole genome alignments and (3) gene trees with protein homology inference. Finally, we show the efficiency of the system in real case scenarios.

Conclusions

eHive allows us to produce computationally demanding results in a reliable and efficient way with minimal supervision and high throughput. Further documentation is available at: http://www.ensembl.org/info/docs/eHive/.

Background

The maturing field of genomics is rapidly increasing the number of sequenced genomes and producing more information from those previously sequenced. Much of this additional information is variation data derived from sampling multiple individuals of a given species with the goal of discovering new variants and characterising the population frequencies of the variants that are already known. These data have immense value for many studies, including those designed to understand evolution and connect genotype to phenotype. Maximising the utility of the data requires that it be stored in an accessible manner that facilitates the integration of variation data with other genome resources such as gene annotation and comparative genomics.

Description

The Ensembl project provides comprehensive and integrated variation resources for a wide variety of chordate genomes. This paper provides a detailed description of the sources of data and the methods for creating the Ensembl variation databases. It also explores the utility of the information by explaining the range of query options available, from using interactive web displays, to online data mining tools and connecting directly to the data servers programmatically. It gives a good overview of the variation resources and future plans for expanding the variation data within Ensembl.

Conclusions

Variation data is an important key to understanding the functional and phenotypic differences between individuals. The development of new sequencing and genotyping technologies is greatly increasing the amount of variation data known for almost all genomes. The Ensembl variation resources are integrated into the Ensembl genome browser and provide a comprehensive way to access this data in the context of a widely used genome bioinformatics system. All Ensembl data is freely available at http://www.ensembl.org and from the public MySQL database server at ensembldb.ensembl.org.

Background

Gene expression arrays are valuable and widely used tools for biomedical research. Today's commercial arrays attempt to measure the expression level of all of the genes in the genome. Effectively translating the results from the microarray into a biological interpretation requires an accurate mapping between the probesets on the array and the genes that they are targeting. Although major array manufacturers provide annotations of their gene expression arrays, the methods used by various manufacturers are different and the annotations are difficult to keep up to date in the rapidly changing world of biological sequence databases.

Results

We have created a consistent microarray annotation protocol applicable to all of the major array manufacturers. We constantly keep our annotations updated with the latest Ensembl Gene predictions, and thus cross-referenced with a large number of external biomedical sequence database identifiers. We show that these annotations are accurate and address in detail reasons for the minority of probesets that cannot be annotated. Annotations are publicly accessible through the Ensembl Genome Browser and programmatically through the Ensembl Application Programming Interface. They are also seamlessly integrated into the BioMart data-mining tool and the biomaRt package of BioConductor.

Conclusions

Consistent, accurate and updated gene expression array annotations remain critical for biological research. Our annotations facilitate accurate biological interpretation of gene expression profiles.