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author:("flick, Paul")
1.  Transcriptome and genome sequencing uncovers functional variation in humans 
Nature  2013;501(7468):506-511.
Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of mRNA and miRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project – the first uniformly processed RNA-seq data from multiple human populations with high-quality genome sequences. We discovered extremely widespread genetic variation affecting regulation of the majority of genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on cellular mechanisms of regulatory and loss-of-function variation, and allowed us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome.
PMCID: PMC3918453  PMID: 24037378
2.  Integrative Annotation of Variants from 1092 Humans: Application to Cancer Genomics 
Science (New York, N.Y.)  2013;342(6154):1235587.
Interpreting variants, especially noncoding ones, in the increasing number of personal genomes is challenging. We used patterns of polymorphisms in functionally annotated regions in 1092 humans to identify deleterious variants; then we experimentally validated candidates. We analyzed both coding and noncoding regions, with the former corroborating the latter. We found regions particularly sensitive to mutations (“ultrasensitive”) and variants that are disruptive because of mechanistic effects on transcription-factor binding (that is, “motif-breakers”). We also found variants in regions with higher network centrality tend to be deleterious. Insertions and deletions followed a similar pattern to single-nucleotide variants, with some notable exceptions (e.g., certain deletions and enhancers). On the basis of these patterns, we developed a computational tool (FunSeq), whose application to ~90 cancer genomes reveals nearly a hundred candidate noncoding drivers.
PMCID: PMC3947637  PMID: 24092746
3.  Ensembl 2014 
Nucleic Acids Research  2013;42(D1):D749-D755.
Ensembl ( creates tools and data resources to facilitate genomic analysis in chordate species with an emphasis on human, major vertebrate model organisms and farm animals. Over the past year we have increased the number of species that we support to 77 and expanded our genome browser with a new scrollable overview and improved variation and phenotype views. We also report updates to our core datasets and improvements to our gene homology relationships from the addition of new species. Our REST service has been extended with additional support for comparative genomics and ontology information. Finally, we provide updated information about our methods for data access and resources for user training.
PMCID: PMC3964975  PMID: 24316576
4.  The NHGRI GWAS Catalog, a curated resource of SNP-trait associations 
Nucleic Acids Research  2013;42(D1):D1001-D1006.
The National Human Genome Research Institute (NHGRI) Catalog of Published Genome-Wide Association Studies (GWAS) Catalog provides a publicly available manually curated collection of published GWAS assaying at least 100 000 single-nucleotide polymorphisms (SNPs) and all SNP-trait associations with P <1 × 10−5. The Catalog includes 1751 curated publications of 11 912 SNPs. In addition to the SNP-trait association data, the Catalog also publishes a quarterly diagram of all SNP-trait associations mapped to the SNPs’ chromosomal locations. The Catalog can be accessed via a tabular web interface, via a dynamic visualization on the human karyotype, as a downloadable tab-delimited file and as an OWL knowledge base. This article presents a number of recent improvements to the Catalog, including novel ways for users to interact with the Catalog and changes to the curation infrastructure.
PMCID: PMC3965119  PMID: 24316577
5.  Locus Reference Genomic: reference sequences for the reporting of clinically relevant sequence variants 
Nucleic Acids Research  2013;42(D1):D873-D878.
Locus Reference Genomic (LRG; records contain internationally recognized stable reference sequences designed specifically for reporting clinically relevant sequence variants. Each LRG is contained within a single file consisting of a stable ‘fixed’ section and a regularly updated ‘updatable’ section. The fixed section contains stable genomic DNA sequence for a genomic region, essential transcripts and proteins for variant reporting and an exon numbering system. The updatable section contains mapping information, annotation of all transcripts and overlapping genes in the region and legacy exon and amino acid numbering systems. LRGs provide a stable framework that is vital for reporting variants, according to Human Genome Variation Society (HGVS) conventions, in genomic DNA, transcript or protein coordinates. To enable translation of information between LRG and genomic coordinates, LRGs include mapping to the human genome assembly. LRGs are compiled and maintained by the National Center for Biotechnology Information (NCBI) and European Bioinformatics Institute (EBI). LRG reference sequences are selected in collaboration with the diagnostic and research communities, locus-specific database curators and mutation consortia. Currently >700 LRGs have been created, of which >400 are publicly available. The aim is to create an LRG for every locus with clinical implications.
PMCID: PMC3965024  PMID: 24285302
6.  The International Mouse Phenotyping Consortium Web Portal, a unified point of access for knockout mice and related phenotyping data 
Nucleic Acids Research  2013;42(D1):D802-D809.
The International Mouse Phenotyping Consortium (IMPC) web portal ( provides the biomedical community with a unified point of access to mutant mice and rich collection of related emerging and existing mouse phenotype data. IMPC mouse clinics worldwide follow rigorous highly structured and standardized protocols for the experimentation, collection and dissemination of data. Dedicated ‘data wranglers’ work with each phenotyping center to collate data and perform quality control of data. An automated statistical analysis pipeline has been developed to identify knockout strains with a significant change in the phenotype parameters. Annotation with biomedical ontologies allows biologists and clinicians to easily find mouse strains with phenotypic traits relevant to their research. Data integration with other resources will provide insights into mammalian gene function and human disease. As phenotype data become available for every gene in the mouse, the IMPC web portal will become an invaluable tool for researchers studying the genetic contributions of genes to human diseases.
PMCID: PMC3964955  PMID: 24194600
7.  Cooperativity and Rapid Evolution of Cobound Transcription Factors in Closely Related Mammals 
Cell  2013;154(3):530-540.
To mechanistically characterize the microevolutionary processes active in altering transcription factor (TF) binding among closely related mammals, we compared the genome-wide binding of three tissue-specific TFs that control liver gene expression in six rodents. Despite an overall fast turnover of TF binding locations between species, we identified thousands of TF regions of highly constrained TF binding intensity. Although individual mutations in bound sequence motifs can influence TF binding, most binding differences occur in the absence of nearby sequence variations. Instead, combinatorial binding was found to be significant for genetic and evolutionary stability; cobound TFs tend to disappear in concert and were sensitive to genetic knockout of partner TFs. The large, qualitative differences in genomic regions bound between closely related mammals, when contrasted with the smaller, quantitative TF binding differences among Drosophila species, illustrate how genome structure and population genetics together shape regulatory evolution.
Graphical Abstract
•Earliest steps of regulatory evolution in mammals captured using five mouse species•Interspecies differences in TF binding are rarely caused by DNA variation in motifs•Cobound TFs change their genomic binding cooperatively in closely related mammals•Genetic knockouts revealed the extent of cooperative stabilization in TF binding clusters
Microevolutionary mechanisms create different transcription factor binding patterns between mammals, shedding light on the regulatory mechanisms partially underlying speciation.
PMCID: PMC3732390  PMID: 23911320
8.  Genome Sequencing Reveals Loci under Artificial Selection that Underlie Disease Phenotypes in the Laboratory Rat 
Cell  2013;154(3):691-703.
Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.
Graphical Abstract
•Genomes of 27 rat strains were sequenced; >13 million sequence variants identified•Selective sweeps and coevolved gene clusters were detected in 11 disease models•Previously identified and new disease genes and pathways were identified•This is first evolutionary analysis of artificial selection for disease phenotypes
Evolution analysis of artificial selection for disease phenotypes, such as hypertension and diabetes, in 27 rat strains reveals disease-related variants and loci.
PMCID: PMC3732391  PMID: 23890820
9.  Latent Regulatory Potential of Human-Specific Repetitive Elements 
Molecular Cell  2013;49(2):262-272.
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.
► 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
PMCID: PMC3560060  PMID: 23246434
10.  Ensembl 2013 
Nucleic Acids Research  2012;41(D1):D48-D55.
The Ensembl project ( 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 and through other tools and programmatic interfaces.
PMCID: PMC3531136  PMID: 23203987
11.  dbVar and DGVa: public archives for genomic structural variation 
Nucleic Acids Research  2012;41(D1):D936-D941.
Much has changed in the last two years at DGVa ( and 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.
PMCID: PMC3531204  PMID: 23193291
12.  The 1000 Genomes Project: Data Management and Community Access 
Nature methods  2012;9(5):459-462.
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.
PMCID: PMC3340611  PMID: 22543379
13.  De novo assembly and genotyping of variants using colored de Bruijn graphs 
Nature genetics  2012;44(2):226-232.
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.
PMCID: PMC3272472  PMID: 22231483
14.  A high-resolution map of human evolutionary constraint using 29 mammals 
Nature  2011;478(7370):476-482.
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.
PMCID: PMC3207357  PMID: 21993624
15.  Considerations for the inclusion of 2x mammalian genomes in phylogenetic analyses 
Genome Biology  2011;12(2):401.
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.
PMCID: PMC3188792  PMID: 21320298
17.  Waves of Retrotransposon Expansion Remodel Genome Organization and CTCF Binding in Multiple Mammalian Lineages 
Cell  2012;148(1-2):335-348.
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.
Graphical Abstract
► 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.
PMCID: PMC3368268  PMID: 22244452
18.  Ensembl 2012 
Nucleic Acids Research  2011;40(D1):D84-D90.
The Ensembl project ( 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; with preliminary support. The past year has also seen improvements across the project.
PMCID: PMC3245178  PMID: 22086963
19.  The functional spectrum of low-frequency coding variation 
Genome Biology  2011;12(9):R84.
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.
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.
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.
PMCID: PMC3308047  PMID: 21917140
21.  Ensembl BioMarts: a hub for data retrieval across taxonomic space 
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:;;;;;
PMCID: PMC3170168  PMID: 21785142
23.  Five vertebrate ChIP-seq reveals the evolutionary dynamics of transcription factor binding 
Science (New York, N.Y.)  2010;328(5981):1036-1040.
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.
PMCID: PMC3008766  PMID: 20378774
24.  Ensembl 2011 
Nucleic Acids Research  2010;39(Database issue):D800-D806.
The Ensembl project ( 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.
PMCID: PMC3013672  PMID: 21045057
25.  Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis 
PLoS Biology  2010;8(9):e1000475.
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.
PMCID: PMC2935454  PMID: 20838655

Results 1-25 (47)