Thomas Derrien

Landscape of transcription in human cells

Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell’s regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene.

The GENCODE v7 catalog of human long noncoding RNAs: Analysis of their gene structure, evolution, and expression

The human genome contains many thousands of long noncoding RNAs (lncRNAs). While several studies have demonstrated compelling biological and disease roles for individual examples, analytical and experimental approaches to investigate these genes have been hampered by the lack of comprehensive lncRNA annotation. Here, we present and analyze the most complete human lncRNA annotation to date, produced by the GENCODE consortium within the framework of the ENCODE project and comprising 9277 manually annotated genes producing 14,880 transcripts. Our analyses indicate that lncRNAs are generated through pathways similar to that of protein-coding genes, with similar histone-modification profiles, splicing signals, and exon/intron lengths. In contrast to protein-coding genes, however, lncRNAs display a striking bias toward two-exon transcripts, they are predominantly localized in the chromatin and nucleus, and a fraction appear to be preferentially processed into small RNAs. They are under stronger selective pressure than neutrally evolving sequences-particularly in their promoter regions, which display levels of selection comparable to protein-coding genes. Importantly, about one-third seem to have arisen within the primate lineage. Comprehensive analysis of their expression in multiple human organs and brain regions shows that lncRNAs are generally lower expressed than protein-coding genes, and display more tissue-specific expression patterns, with a large fraction of tissue-specific lncRNAs expressed in the brain. Expression correlation analysis indicates that lncRNAs show particularly striking positive correlation with the expression of antisense coding genes. This GENCODE annotation represents a valuable resource for future studies of lncRNAs.

Long noncoding RNAs with enhancer-like function in human cells

While the long noncoding RNAs (ncRNAs) constitute a large portion of the mammalian transcriptome, their biological functions has remained elusive. A few long ncRNAs that have been studied in any detail silence gene expression in processes such as X-inactivation and imprinting. We used a GENCODE annotation of the human genome to characterize over a thousand long ncRNAs that are expressed in multiple cell lines. Unexpectedly, we found an enhancer-like function for a set of these long ncRNAs in human cell lines. Depletion of a number of ncRNAs led to decreased expression of their neighboring protein-coding genes, including the master regulator of hematopoiesis, SCL (also called TAL1), Snai1 and Snai2. Using heterologous transcription assays we demonstrated a requirement for the ncRNAs in activation of gene expression. These results reveal an unanticipated role for a class of long ncRNAs in activation of critical regulators of development and differentiation.

Fast computation and applications of genome mappability.

We present a fast mapping-based algorithm to compute the mappability of each region of a reference genome up to a specified number of mismatches. Knowing the mappability of a genome is crucial for the interpretation of massively parallel sequencing experiments. We investigate the properties of the mappability of eukaryotic DNA/RNA both as a whole and at the level of the gene family, providing for various organisms tracks which allow the mappability information to be visually explored. In addition, we show that mappability varies greatly between species and gene classes. Finally, we suggest several practical applications where mappability can be used to refine the analysis of high-throughput sequencing data (SNP calling, gene expression quantification and paired-end experiments). This work highlights mappability as an important concept which deserves to be taken into full account, in particular when massively parallel sequencing technologies are employed. The GEM mappability program belongs to the GEM (GEnome Multitool) suite of programs, which can be freely downloaded for any use from its website (http://gemlibrary.sourceforge.net).

Identification of genomic regions associated with phenotypic variation between dog breeds using selection mapping.

The extraordinary phenotypic diversity of dog breeds has been sculpted by a unique population history accompanied by selection for novel and desirable traits. Here we perform a comprehensive analysis using multiple test statistics to identify regions under selection in 509 dogs from 46 diverse breeds using a newly developed high-density genotyping array consisting of >170,000 evenly spaced SNPs. We first identify 44 genomic regions exhibiting extreme differentiation across multiple breeds. Genetic variation in these regions correlates with variation in several phenotypic traits that vary between breeds, and we identify novel associations with both morphological and behavioral traits. We next scan the genome for signatures of selective sweeps in single breeds, characterized by long regions of reduced heterozygosity and fixation of extended haplotypes. These scans identify hundreds of regions, including 22 blocks of homozygosity longer than one megabase in certain breeds. Candidate selection loci are strongly enriched for developmental genes. We chose one highly differentiated region, associated with body size and ear morphology, and characterized it using high-throughput sequencing to provide a list of variants that may directly affect these traits. This study provides a catalogue of genomic regions showing extreme reduction in genetic variation or population differentiation in dogs, including many linked to phenotypic variation. The many blocks of reduced haplotype diversity observed across the genome in dog breeds are the result of both selection and genetic drift, but extended blocks of homozygosity on a megabase scale appear to be best explained by selection. Further elucidation of the variants under selection will help to uncover the genetic basis of complex traits and disease.

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.

The Long Non-Coding RNAs: A New (P)layer in the “Dark Matter”

The transcriptome of a cell is represented by a myriad of different RNA molecules with and without protein-coding capacities. In recent years, advances in sequencing technologies have allowed researchers to more fully appreciate the complexity of whole transcriptomes, showing that the vast majority of the genome is transcribed, producing a diverse population of non-protein coding RNAs (ncRNAs). Thus, the biological significance of non-coding RNAs (ncRNAs) have been largely underestimated. Amongst these multiple classes of ncRNAs, the long non-coding RNAs (lncRNAs) are apparently the most numerous and functionally diverse. A small but growing number of lncRNAs have been experimentally studied, and a view is emerging that these are key regulators of epigenetic gene regulation in mammalian cells. LncRNAs have already been implicated in human diseases such as cancer and neurodegeneration, highlighting the importance of this emergent field. In this article, we review the catalogs of annotated lncRNAs and the latest advances in our understanding of lncRNAs.

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.

Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome

Within the ENCODE Consortium, GENCODE aimed to accurately annotate all protein-coding genes, pseudogenes, and noncoding transcribed loci in the human genome through manual curation and computational methods. Annotated transcript structures were assessed, and less well-supported loci were systematically, experimentally validated. Predicted exon-exon junctions were evaluated by RT-PCR amplification followed by highly multiplexed sequencing readout, a method we called RT-PCR-seq. Seventy-nine percent of all assessed junctions are confirmed by this evaluation procedure, demonstrating the high quality of the GENCODE gene set. RT-PCR-seq was also efficient to screen gene models predicted using the Human Body Map (HBM) RNA-seq data. We validated 73% of these predictions, thus confirming 1168 novel genes, mostly noncoding, which will further complement the GENCODE annotation. Our novel experimental validation pipeline is extremely sensitive, far more than unbiased transcriptome profiling through RNA sequencing, which is becoming the norm. For example, exon-exon junctions unique to GENCODE annotated transcripts are five times more likely to be corroborated with our targeted approach than with extensive large human transcriptome profiling. Data sets such as the HBM and ENCODE RNA-seq data fail sampling of low-expressed transcripts. Our RT-PCR-seq targeted approach also has the advantage of identifying novel exons of known genes, as we discovered unannotated exons in ~11% of assessed introns. We thus estimate that at least 18% of known loci have yet-unannotated exons. Our work demonstrates that the cataloging of all of the genic elements encoded in the human genome will necessitate a coordinated effort between unbiased and targeted approaches, like RNA-seq and RT-PCR-seq.

AutoGRAPH: an interactive web server for automating and visualizing comparative genome maps

UNLABELLED AutoGRAPH is an interactive web server for automatic multi-species comparative genomics analyses based on personal datasets or pre-inserted public datasets. This program automatically identifies conserved segments (CS) and breakpoint regions, assesses the conservation of marker/gene order between organisms, constructs synteny maps for two to three species and generates high-quality, interactive displays facilitating the identification of chromosomal rearrangements. AutoGRAPH can also be used for the integration and comparison of several types of genomic resources (meiotic maps, radiation hybrid maps and genome sequences) for a single species, making AutoGRAPH a versatile tool for comparative genomics analysis. AVAILABILITY http://genoweb.univ-rennes1.fr/tom_dog/AutoGRAPH/. SUPPLEMENTARY INFORMATION A description of the algorithm and additional information are available at http://genoweb.univ-rennes1.fr/tom_dog/AutoGRAPH/Tutorial.php.