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Within the scope of the FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem cells (iPSCs) and systematically characterize their roles in self-renewal and pluripotency. We find 36 lncRNAs (18%) exhibiting cell growth inhibition. From the knockdown of 123 lncRNAs with transcriptome profiling, 36 lncRNAs (29.3%) show molecular phenotypes. Integrating the molecular phenotypes with chromatin-interaction assays further reveals cis- and trans-interacting partners as potential primary targets. Additionally, cell-type enrichment analysis identifies lncRNAs associated with pluripotency, while the knockdown of LINC02595, CATG00000090305.1, and RP11-148B6.2 modulates colony formation of iPSCs. We compare our results with previously published fibroblasts phenotyping data and find that 2.9% of the lncRNAs exhibit a consistent cell growth phenotype, whereas we observe 58.3% agreement in molecular phenotypes. This highlights that molecular phenotyping is more comprehensive in revealing affected pathways.

Gene expression profiles in homologous tissues have been observed to be different between species, which may be due to differences between species in the gene expression program in each cell type, but may also reflect differences in cell type composition of each tissue in different species. Here, we compare expression profiles in matching primary cells in human, mouse, rat, dog, and chicken using Cap Analysis Gene Expression (CAGE) and short RNA (sRNA) sequencing data from FANTOM5. While we find that expression profiles of orthologous genes in different species are highly correlated across cell types, in each cell type many genes were differentially expressed between species. Expression of genes with products involved in transcription, RNA processing, and transcriptional regulation was more likely to be conserved, while expression of genes encoding proteins involved in intercellular communication was more likely to have diverged during evolution. Conservation of expression correlated positively with the evolutionary age of genes, suggesting that divergence in expression levels of genes critical for cell function was restricted during evolution. Motif activity analysis showed that both promoters and enhancers are activated by the same transcription factors in different species. An analysis of expression levels of mature miRNAs and of primary miRNAs identified by CAGE revealed that evolutionary old miRNAs are more likely to have conserved expression patterns than young miRNAs. We conclude that key aspects of the regulatory network are conserved, while differential expression of genes involved in cell-to-cell communication may contribute greatly to phenotypic differences between species.

BackgroundUsing the Cap Analysis of Gene Expression technology, the FANTOM5 consortium provided one of the most comprehensive maps of Transcription Start Sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers.ResultsHere, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at short tandem repeats (STRs) corresponding to homopolymers of thymidines (T). Additional analyse confirm that these CAGEs are truly associated with transcriptionally active chromatin marks. Furthermore, we train a sequence-based deep learning model able to predict CAGE signal at T STRs with high accuracy (~81%) Extracting features learned by this model reveals that transcription at T STRs is mostly directed by STR length but also instructions lying in the downstream sequence. Excitingly, our model also predicts that genetic variants linked to human diseases affect this STR-associated transcription.ConclusionsTogether, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism. We also provide a new metric that can be considered in future studies of STR-related complex traits.

Non-allelic recombination between homologous repetitive elements contributes to evolution and human genetic disorders. Here, we combine short- and long-DNA read sequencing of repeat elements with a new bioinformatics pipeline to show that somatic recombination of Alu and L1 elements is widespread in the human genome. Our analysis uncovers tissue-specific non-allelic homologous recombination hallmarks; moreover, we find that centromeres and cancer-associated genes are enriched for retroelements that may act as recombination hotspots. We compare recombination profiles in human-induced pluripotent stem cells and differentiated neurons and find that the neuron-specific recombination of repeat elements accompanies chromatin changes during cell-fate determination. Finally, we report that somatic recombination profiles are altered in Parkinson's and Alzheimer's disease, suggesting a link between retroelement recombination and genomic instability in neurodegeneration. This work highlights a significant contribution of the somatic recombination of repeat elements to genomic diversity in health and disease.

Profiling of cis-regulatory elements (CREs, mostly promoters and enhancers) in single cells allows the interrogation of the cell-type and cell-state-specific contexts of gene regulation and genetic predisposition to diseases. Here we demonstrate single-cell RNA-5′end-sequencing (sc-end5-seq) methods can detect transcribed CREs (tCREs), enabling simultaneous quantification of gene expression and enhancer activities in a single assay at no extra cost. We showed enhancer RNAs can be detected using sc-end5-seq methods with either random or oligo(dT) priming. To analyze tCREs in single cells, we developed SCAFE (Single Cell Analysis of Five-prime Ends) to identify genuine tCREs and analyze their activities (https://github.com/chung-lab/scafe). As compared to accessible CRE (aCRE, based on chromatin accessibility), tCREs are more accurate in predicting CRE interactions by co-activity, more sensitive in detecting shifts in alternative promoter usage and more enriched in diseases heritability. Our results highlight additional dimensions within sc-end5-seq data which can be used for interrogating gene regulation and disease heritability.

The human genome is pervasively transcribed and produces a wide variety of long non-coding RNAs (lncRNAs), constituting the majority of transcripts across human cell types. Studying lncRNAs is challenging due to their low expression level, cell type-specific occurrence, poor sequence conservation between orthologs, and lack of information about RNA domains. LncRNAs direct the regulatory factors in the locations that are in cis to their transcription sites. We designed a model to predict if an lncRNA acts in cis based on its features and trained it using RNA-chromatin interaction data. The trained model is cell type-independent and does not require RNA-chromatin data. Combining RNA-chromatin and Hi-C data, we showed that lncRNA-chromatin binding sites are determined by chromosome conformation. For each lncRNA, the spatially proximal genes were identified as their potential targets by combining Hi-C and Cap Analysis Gene Expression (CAGE) data in 18 human cell types. RNA-protein and RNA-chromatin interaction data suggested that lncRNAs act as scaffolds to recruit regulatory proteins to target promoters and enhancers. We provide the data through an interactive visualization web portal at https://fantom.gsc.riken.jp/zenbu/reports/#F6_3D_lncRNA.

SummaryThe recombination of millions of Alu and L1 copies in our genomes contributes to evolution and genetic disorders, but the somatic extent of these rearrangements has not been systematically investigated. Here we combine short and long-reads reads DNA sequencing with a new bioinformatic pipeline to show that somatic recombination of Alu and L1 elements is common in human genomes. We report new tissue-specific recombination hallmarks, and show that retroelements acting as recombination hotspots are enriched in centromeres and cancer genes. We compare recombination profiles in induced pluripotent stem cells and differentiated neurons and show that neuron-specific recombination of repeat elements accompanies chromatin reorganization during cell-fate determination. Finally, we find that somatic recombination profiles are altered in Parkinson’s and Alzheimer’s disease, indicating a link between retroelements recombination and genomic instability in neurodegeneration. This work shows that somatic recombination of repeat elements contributes massively to genomic diversity in health and disease.

SummaryMillions of Alu and L1 copies in our genomes contribute to evolution and genetic disorders via non-allelic homologous recombination, but the somatic extent of these rearrangements has not been systematically investigated. Here we combine short and long DNA reads sequencing of repeat elements with a new bioinformatic pipeline to show that somatic recombination of Alu and L1 elements is common in human genomes. We report new tissue-specific recombination hallmarks, and show that retroelements acting as recombination hotspots are enriched in centromeres and cancer genes. We compare recombination profiles in human induced pluripotent stem cells and differentiated neurons and show that neuron-specific recombination of repeat elements accompanies chromatin changes during cell-fate determination. Finally, we find that somatic recombination profiles are altered in Parkinson’s and Alzheimer’s disease, indicating a link between retroelements recombination and genomic instability in neurodegeneration. This work shows that somatic recombination of repeat elements contributes massively to genomic diversity in health and disease.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Aggressive therapy-resistant and refractory acute myeloid leukemia (AML) has an extremely poor outcome. By analyzing a large number of genetically complex and diverse, primary high-risk poor-outcome human AML samples, we identified specific pathways of therapeutic vulnerability. Through drug screens followed by extensive in vivo validation and genomic analyses, we found inhibition of cytosolic and mitochondrial anti-apoptotic proteins XIAP, BCL2 and MCL1, and a key regulator of mitosis, AURKB, as a vulnerability hub based on patient-specific genetic aberrations and transcriptional signatures. Combinatorial therapeutic inhibition of XIAP with an additional patient-specific vulnerability eliminated established AML in vivo in patient-derived xenografts (PDXs) bearing diverse genetic aberrations, with no signs of recurrence during off-treatment follow-up. By integrating genomic profiling and drug-sensitivity testing, this work provides a platform for a precision-medicine approach for treating aggressive AML with high unmet need. Ishikawa and colleagues perform integrated genomic and drug-sensitivity screens with extensive PDX modeling and reveal combined XIAP and BCL2 inhibition as a vulnerability hub across AML genetic alterations.

The FANTOM web resource (http://fantom.gsc.riken.jp/) was developed to provide easy access to the data produced by the FANTOM project. It contains the most complete and comprehensive sets of actively transcribed enhancers and promoters in the human and mouse genomes. We determined the transcription activities of these regulatory elements by CAGE (Cap Analysis of Gene Expression) for both steady and dynamic cellular states in all major and some rare cell types, consecutive stages of differentiation and responses to stimuli. We have expanded the resource by employing different assays, such as RNA-seq, short RNA-seq and a paired-end protocol for CAGE (CAGEscan), to provide new angles to study the transcriptome. That yielded additional atlases of long noncoding RNAs, miRNAs and their promoters. We have also expanded the CAGE analysis to cover rat, dog, chicken, and macaque species for a limited number of cell types. The CAGE data obtained from human and mouse were reprocessed to make them available on the latest genome assemblies. Here, we report the recent updates of both data and interfaces in the FANTOM web resource.

The response of the human acute myeloid leukemia cell line THP-1 to phorbol esters has been widely-studied to test candidateleukemia therapies and as a model of cell cycle arrest and monocyte-macrophage differentiation. Here we have employed CapAnalysis of Gene Expression (CAGE) to analyse a dense time course of transcriptional regulation in THP-1 cells treated with phorbolmyristate acetate (PMA) over 96 hours. PMA treatment greatly reduced the numbers of cells entering S phase and also blockedcells exiting G2/M. The PMA-treated cells became adherent and expression of mature macrophage-specific genes increasedprogressively over the duration of the time course. Within 1-2 hours PMA induced known targets of tumour protein p53 (TP53),notably CDKN1A, followed by gradual down-regulation of cell-cycle associated genes. Also within the first 2 hours, PMA inducedimmediate early genes including transcription factor genes encoding proteins implicated in macrophage differentiation (EGR2, JUN,MAFB) and down-regulated genes for transcription factors involved in immature myeloid cell proliferation (MYB, IRF8, GFI1). Thedense time course revealed that the response to PMA was not linear and progressive. Rather, network-based clustering of thetime course data highlighted a sequential cascade of transient up- and down-regulated expression of genes encoding feedbackregulators, as well as transcription factors associated with macrophage differentiation and their inferred target genes. CAGEalso identified known and candidate novel enhancers expressed in THP-1 cells and many novel inducible genes that currently lackfunctional annotation and/or had no previously known function in macrophages. The time course is available on the ZENBUplatform allowing comparison to FANTOM4 and FANTOM5 data.

Long non-coding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes and yet, their functions remain largely unknown. We systematically knockdown 285 lncRNAs expression in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNA exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest to-date lncRNA knockdown dataset with molecular phenotyping (over 1,000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Rhesus macaque was the second non-human primate whose genome has been fully sequenced and is one of the most used model organisms to study human biology and disease, thanks to the close evolutionary relationship between the two species. But compared to human, where several previously unknown RNAs have been uncovered, the macaque transcriptome is less studied. Publicly available RNA expression resources for macaque are limited, even for brain, which is highly relevant to study human cognitive abilities. In an effort to complement those resources, FANTOM5 profiled 15 distinct anatomical regions of the aged macaque central nervous system using Cap Analysis of Gene Expression, a high-resolution, annotation-independent technology that allows monitoring of transcription initiation events with high accuracy. We identified 25,869 CAGE peaks, representing bona fide promoters. For each peak we provide detailed annotation, expanding the landscape of ‘known’ macaque genes, and we show concrete examples on how to use the resulting data. We believe this data represents a useful resource to understand the central nervous system in macaque.

Upon the first publication of the fifth iteration of the Functional Annotation of Mammalian Genomes collaborative project, FANTOM5, we gathered a series of primary data and database systems into the FANTOM web resource (http://fantom.gsc.riken.jp) to facilitate researchers to explore transcriptional regulation and cellular states. In the course of the collaboration, primary data and analysis results have been expanded, and functionalities of the database systems enhanced. We believe that our data and web systems are invaluable resources, and we think the scientific community will benefit for this recent update to deepen their understanding of mammalian cellular organization. We introduce the contents of FANTOM5 here, report recent updates in the web resource and provide future perspectives. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/)

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3′-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5′-ends with an original sample multiplexing strategy in the C1TM microfluidic system. We first quantifiy the performance of C1 CAGE and find it as accurate and sensitive as other methods in the C1 system. We then use it to profile promoter and enhancer activities in the cellular response to TGF-β of lung cancer cells and discover subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand in a mutually exclusive manner, validated using single molecule fluorescence in situ hybridization., Single-cell transcriptomic profiling allows the exploration of cellular heterogeneity but commonly focuses on the 3′-end of the transcript. Here the authors introduce C1 CAGE, which detects the 5′ transcript end in a multiplexed microfluidic system.

Uncaging promoter and enhancer dynamics In order to understand cellular differentiation, it is important to understand the timing of the regulation of gene expression. Arner et al. used cap analysis of gene expression (CAGE) to analyze gene enhancer and promoter activities in a number of human and mouse cell types. The RNA of enhancers was transcribed first, followed by that of transcription factors, and finally by genes that are not transcription factors. Science , this issue p. 1010

The promoter landscape of several non-human model organisms is far from complete. As a part of FANTOM5 data collection, we generated 13 profiles of transcription initiation activities in dog and rat aortic smooth muscle cells, mesenchymal stem cells and hepatocytes by employing CAGE (Cap Analysis of Gene Expression) technology combined with single molecule sequencing. Our analyses show that the CAGE profiles recapitulate known transcription start sites (TSSs) consistently, in addition to uncover novel TSSs. Our dataset can be thus used with high confidence to support gene annotation in dog and rat species. We identified 28,497 and 23,147 CAGE peaks, or promoter regions, for rat and dog respectively, and associated them to known genes. This approach could be seen as a standard method for improvement of existing gene models, as well as discovery of novel genes. Given that the FANTOM5 data collection includes dog and rat matched cell types in human and mouse as well, this data would also be useful for cross-species studies.

The FANTOM5 expression atlas is a quantitative measurement of the activity of nearly 200,000 promoter regions across nearly 2,000 different human primary cells, tissue types and cell lines. Generation of this atlas was made possible by the use of CAGE, an experimental approach to localise transcription start sites at single-nucleotide resolution by sequencing the 5′ ends of capped RNAs after their conversion to cDNAs. While 50% of CAGE-defined promoter regions could be confidently associated to adjacent transcriptional units, nearly 100,000 promoter regions remained gene-orphan. To address this, we used the CAGEscan method, in which random-primed 5′-cDNAs are paired-end sequenced. Pairs starting in the same region are assembled in transcript models called CAGEscan clusters. Here, we present the production and quality control of CAGEscan libraries from 56 FANTOM5 RNA sources, which enhances the FANTOM5 expression atlas by providing experimental evidence associating core promoter regions with their cognate transcripts.

Scientific Data, 4, ISSN:2052-4463

The FANTOM5 consortium described the promoter-level expression atlas of human and mouse by using CAGE (Cap Analysis of Gene Expression) with single molecule sequencing. In the original publications, GRCh37/hg19 and NCBI37/mm9 assemblies were used as the reference genomes of human and mouse respectively; later, the Genome Reference Consortium released newer genome assemblies GRCh38/hg38 and GRCm38/mm10. To increase the utility of the atlas in forthcoming researches, we reprocessed the data to make them available on the recent genome assemblies. The data include observed frequencies of transcription starting sites (TSSs) based on the realignment of CAGE reads, and TSS peaks that are converted from those based on the previous reference. Annotations of the peak names were also updated based on the latest public databases. The reprocessed results enable us to examine frequencies of transcription initiations on the recent genome assemblies and to refer promoters with updated information across the genome assemblies consistently.

Rhesus macaque was the second non-human primate whose genome has been fully sequenced and is one of the most used model organisms to study human biology and disease, thanks to the close evolutionary relationship between the two species. But compared to human, where several previously unknown RNAs have been uncovered, the macaque transcriptome is less studied. Publicly available RNA expression resources for macaque are limited, even for brain, which is highly relevant to study human cognitive abilities. In an effort to complement those resources, FANTOM5 profiled 15 distinct anatomical regions of the aged macaque central nervous system using Cap Analysis of Gene Expression, a high-resolution, annotation-independent technology that allows monitoring of transcription initiation events with high accuracy. We identified 25,869 CAGE peaks, representing bona fide promoters. For each peak we provide detailed annotation, expanding the landscape of ‘known’ macaque genes, and we show concrete examples on how to use the resulting data. We believe this data represents a useful resource to understand the central nervous system in macaque. Machine-accessible metadata file describing the reported data (ISA-Tab format)

Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and non-coding RNAs with capped 5′ ends that vary in size. Methods that identify the 5′ ends of transcripts will facilitate the discovery of novel promoters and 5′ ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we have developed nanoCAGE (Cap Analysis of Gene Expression), a method that captures the 5′ ends of transcripts from as little as 10 nanograms of total RNA and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5′ ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome.

Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process.

The use of phylogenetic trees to describe the evolution of biological processes was established in the 1950s (Hennig 1952) and remains a fundamental approach to understanding the evolution of individual genes through to complete genomes; for example, in the mouse (Mouse Genome Sequencing Consortium 2002), rat (Gibbs et al. 2004), chicken (International Chicken Genome Sequencing Consortium 2004), and monodelphis (Mikkelsen et al. 2007) genome papers, and numerous papers on individual sequences. Now routine, the determination of vertebrate genome sequences provides a rich data source to understand evolution, and using phylogenetic trees of the genes is one of the best ways to organize these data. However, the increased set of genomes makes the compute and engineering tasks to form all the gene trees progressively more complex and harder for individual groups to use. The Ensembl project provides an accurate and consistent protein-coding gene set for all vertebrate genomes (International Human Genome Sequencing Consortium 2001; Dehal et al. 2002; Mouse Genome Sequencing Consortium 2002; Gibbs et al. 2004; Xie et al. 2005; Mikkelsen et al. 2007; Rhesus Macaque Genome Sequencing and Analysis Consortium 2007). Previously (until April 2006), Ensembl provided a basic method for tracing orthologs via the Best Reciprocal BLAST method, similar to approaches used in other genome analyses, such as Drosophila melanogaster (Adams et al. 2000) or human (International Human Genome Sequencing Consortium 2001). In June 2006 (Hubbard et al. 2007), we replaced this system with a phylogenetically sound, gene tree-based approach, providing a complete set of phylogenetic trees spanning 91% of genes across vertebrates. In addition to the vertebrates we have included a few important non-vertebrate species (fly, worm, and yeast) to act both as out groups and provide links to these model organisms. In this paper we provide the motivation, implementation, and benchmarking of this method and document the display and access methods for these trees. There have been a number of methods proposed for routine generation of genomewide orthology descriptions, including Inparanoid (Remm et al. 2001), MSOAR (Fu et al. 2007), OrthoMCL (Li et al. 2003), HomoloGene (Wheeler et al. 2008), TreeFam (Li et al. 2006), PhyOP (Goodstadt and Ponting 2006), and PhiGs (Dehal and Boore 2006). The first four, Inparanoid, MSOAR, OrthoMCL, and HomoloGene, focus on providing clusters (or linked clusters) of genes, without an explicit tree topology. PhyOP (Goodstadt and Ponting 2006) uses a tree-based method, but between pairs of closely related species, resolving paralogs accurately by using neutral substitution (as measured by d S, the synonymous substitution rate). TreeFam provides an explicit gene tree across multiple species, using both d S, d N (nonsynonymous substitution rate), nucleotide and protein distance measures, and the standard species tree to balance duplications vs. deletions to inform the tree construction, using the program TreeBeST (http://treesoft.sourceforge.net/treebest.shtml; L. Heng, A.J. Vilella, E. Birney, and R. Durbin, in prep.). The PhiGs method (Dehal and Boore 2006) is a leading phylogenetic-based method that produced a comprehensive phylogenetic resource for the genomes at the time it was run, and the basic outline of its analysis, which was clustering of protein sequences, followed by phylogenetic trees, is similar to the method presented here. However, the PhiGs resource covered a smaller number of species (23 vs. 45) and has been difficult to keep up to date with the advances in gene sets and genomes. Another major difference between PhiG-based phylogenetic trees and the phylogenetic trees presented here is that the former was calculated using a single maximum likelihood method based on protein evolution. In contrast, the Ensembl gene trees are calculated using a new method, TreeBeST, which integrates multiple tree topologies, in particular both DNA level and protein level models and combines this with a species-tree aware penalization of topologies, which are inconsistent with known species relationships. We show in this paper that this method produces trees that are more consistent with synteny relationships and less anomalous topologies than single protein-based phylogenetic methods. There are also many single phylogenetic tree-building approaches, many of them based on maximum likelihood methods; one leading method is PhyML (Guindon and Gascuel 2003). It is unclear what is the best method to use, in particular in the context of genome-wide tree building with constraints on computational costs and the need to robustly handle many complex scenarios usually involving large families with heterogeneous phylogenetic depths. In this paper, we benchmark in vertebrates the tree programs TreeBeST and PhyML, and the resulting trees to basic best reciprocal hit (BRH) methods, and cluster frameworks, in particular Inparanoid and HomoloGene. We also benchmark to a recent PhyOP data set. The PhyOP pipeline has recently switched to use the same tree-building program (TreeBeST) that we use, but differs in its input clusters. Although we adopted this same tree-building method, we describe here considerable novel engineering in the deployment of these methods across all vertebrates. Similar to the PhiGs resource, we have used the dense coverage of genomes to provide topologically based timings (i.e., the standard use of outgroups vs. subsequent lineages to bracket a duplication), in order to label duplication events.

The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). This resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0560-6) contains supplementary material, which is available to authorized users.

Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.

Although female common marmosets typically do not breed while housed with their natal families, up to half ovulate at least once while housed with the intact natal family, and a similar proportion conceive if an unrelated adult male is present in the group. In this study, we investigated the behavioral and social correlates of escape from suppression of ovulation by daughters housed in intact natal families or in families in which the father had been replaced by an unrelated adult male. Focal-animal behavioral data were collected from daughters that were (N = 7) or were not (N = 10) undergoing ovulatory cycles while housed with the natal family and from daughters that were (N = 5) or were not (N = 3) cycling or pregnant in families containing an unrelated male. Additionally, four cyclic and six acyclic females housed in intact natal families underwent simulated "prospecting" tests. Cyclic and acyclic daughters in intact natal families did not engage in sexual interactions with the father and showed few differences from one another in their interactions with the parents. Moreover, cyclic and acyclic daughters did not differ in their willingness to leave the family for short periods or to investigate an unfamiliar family in "prospecting" tests. However, daughters that underwent ovarian cycles in the presence of an unrelated male showed numerous behavioral differences from those in intact natal families, including frequent courtship and sexual behaviors with the male, reduced affiliative interactions with the mother, and elevated frequencies of aggressive display behavior. Moreover, these females were less likely to behave submissively towards the mother or the adult male. These findings suggest that both suppression of ovulation and inhibition of sexual behavior normally contribute to reproductive failure in female marmosets living with their natal families, and that the two components of suppression may become dissociated under specific social conditions.

The fibrillins and latent transforming growth factor binding proteins (LTBPs) form a superfamily of extracellular matrix (ECM) proteins characterized by the presence of a unique domain, the 8-cysteine transforming growth factor beta (TGFβ) binding domain. These proteins are involved in the structure of the extracellular matrix and controlling the bioavailability of TGFβ family members. Genes encoding these proteins show differential expression in mesenchymal cell types which synthesize the extracellular matrix. We have investigated the promoter regions of the seven gene family members using the FANTOM5 CAGE database for human. While the protein and nucleotide sequences show considerable sequence similarity, the promoter regions were quite diverse. Most genes had a single predominant transcription start site region but LTBP1 and LTBP4 had two regions initiating different transcripts. Most of the family members were expressed in a range of mesenchymal and other cell types, often associated with use of alternative promoters or transcription start sites within a promoter in different cell types. FBN3 was the lowest expressed gene, and was found only in embryonic and fetal tissues. The different promoters for one gene were more similar to each other in expression than to promoters of the other family members. Notably expression of all 22 LTBP2 promoters was tightly correlated and quite distinct from all other family members. We located candidate enhancer regions likely to be involved in expression of the genes. Each gene was associated with a unique subset of transcription factors across multiple promoters although several motifs including MAZ, SP1, GTF2I and KLF4 showed overrepresentation across the gene family. FBN1 and FBN2, which had similar expression patterns, were regulated by different transcription factors. This study highlights the role of alternative transcription start sites in regulating the tissue specificity of closely related genes and suggests that this important class of extracellular matrix proteins is subject to subtle regulatory variations that explain the differential roles of members of this gene family., Highlights • We examine expression, promoter use and enhancers for the fibrillin/LTBP gene family. • Promoter switching was observed for most family members. • Multiple enhancers were identified for all family members. • Family members overlapped in tissue specificity with some unique expression patterns. • A degree of redundancy among family members is possible.

The four dye fluorescence detection strategy is a widely used approach to automated DNA sequence analysis. An important aspect of data processing in this approach is the multicomponent analysis to deduce the concentrations of four fluorophores from fluorescence emission intensities at four different wave-lengths. This requires knowledge of the correct transformation matrix M. The matrix M is a function both of the fluorophores employed and the fluorescence detection system. M is typically determined either by a calibration process with individual dyes, or by choosing four well-separated individual peaks corresponding to the four different dyes. Both are time-consuming and complicated procedures for routine use. An automatic scheme for finding M directly from raw sequence data is presented here. This facilitates data analysis and the underlying algorithm may also find utility in other multispectral applications.

Transcriptional regulatory networks (TRN) control the underlying mechanisms behind cellular functions and they are defined by a set of core transcription factors regulating cascades of peripheral genes. Here we report SPI1, CEBPA, MNDA and IRF8 as core transcription factors of monocyte TRN and demonstrate functional inductions of phagocytosis, inflammatory response and chemotaxis activities in human dermal fibroblasts. The Gene Ontology and KEGG pathway analyses also revealed notable representation of genes involved in immune response and endocytosis in fibroblasts. Moreover, monocyte TRN-inducers triggered multiple monocyte-specific genes based on the transcription factor motif response analysis and suggest that complex cellular TRNs are uniquely amenable to elicit cell-specific functions in unrelated cell types.

Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR geneOlfr160(M72) can drive OSN-specific expression in transgenic mice.

The international Functional Annotation Of the Mammalian Genomes 4 (FANTOM4) research collaboration set out to better understand the transcriptional network that regulates macrophage differentiation and to uncover novel components of the transcriptome employing a series of high-throughput experiments. The primary and unique technique is cap analysis of gene expression (CAGE), sequencing mRNA 5′-ends with a second-generation sequencer to quantify promoter activities even in the absence of gene annotation. Additional genome-wide experiments complement the setup including short RNA sequencing, microarray gene expression profiling on large-scale perturbation experiments and ChIP–chip for epigenetic marks and transcription factors. All the experiments are performed in a differentiation time course of the THP-1 human leukemic cell line. Furthermore, we performed a large-scale mammalian two-hybrid (M2H) assay between transcription factors and monitored their expression profile across human and mouse tissues with qRT-PCR to address combinatorial effects of regulation by transcription factors. These interdependent data have been analyzed individually and in combination with each other and are published in related but distinct papers. We provide all data together with systematic annotation in an integrated view as resource for the scientific community (http://fantom.gsc.riken.jp/4/). Additionally, we assembled a rich set of derived analysis results including published predicted and validated regulatory interactions. Here we introduce the resource and its update after the initial release.

BackgroundThe 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.ResultsWe 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.ConclusionseHive 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/.

EdgeExpressDB is a novel database and set of interfaces for interpreting biological networks and comparing large high-throughput expression datasets., EdgeExpressDB is a novel database and set of interfaces for interpreting biological networks and comparing large high-throughput expression datasets that requires minimal development for new data types and search patterns. The FANTOM4 EdgeExpress database summarizes gene expression patterns in the context of alternative promoter structures and regulatory transcription factors and microRNAs using intuitive gene-centric and sub-network views. This is an important resource for gene regulation in acute myeloid leukemia, monocyte/macrophage differentiation and human transcriptional networks.

Modern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species. To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them. Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery. Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species. Since whole-genome optical maps are ordered restriction maps, sequenced strains ofShigella flexneriserotype 2a (2457T and 301),Yersinia pestis(CO 92 and KIM), andEscherichia coliwere aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequencedShigella flexneristrain (serotype Y strain AMC[328Y]) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions. Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.

Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps ( Xho I and Pvu II) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the Xho I map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the Pvu II map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput are enabling such maps to guide sequence assembly at a very early stage of a microbial sequencing project.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

Software for gel image analysis and base-calling in fluorescence-based sequencing consisting of two primary programs, BaseFinder and GelImager, is described. BaseFinder is a framework for trace processing, analysis, and base-calling. BaseFinder is highly extensible, allowing the addition of trace analysis and processing modules without recompilation. Powerful scripting capabilities combined with modularity and multilane handling allow the user to customize BaseFinder to virtually any type of trace processing. We have developed an extensive set of data processing and analysis modules for use with the program in fluorescence-based sequencing. GelImager is a framework for gel image manipulation. It can be used for gel visualization, lane retracking, and as a front end to the Washington University Getlanes program. The programs were designed using a cross-platform development environment, currently allowing them to run in Windows NT, Windows 95, Openstep/Mach, and Rhapsody. Work is ongoing to deploy the software on additional platforms, including Solaris, Linux, and MacOS. This software has been thoroughly tested and debugged in the analysis of >2 million bp of raw sequence data from human chromosome 19 region q13. Overall sequencing accuracy was measured using a significant subset of these data, consisting of ∼600 sequences, by comparing the individual shotgun sequences against the final assembled contigs. Also, results are reported from experiments that analyzed the accuracy of the software and two other well-known base-calling programs for sequencing the M13mp18 vector sequence.[The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF025422]

A modified vector, M13-102, is described which utilizes the previously reported M13-100 direct selection strategy for shotgun cloning [Guilfoyle and Smith, Nucleic Acids Res. 22 (1994) 100-107]. In these vectors, direct selection replaces the need for phosphatase treatment of vector DNA and is achieved by insertional inactivation of M13 gene X. When not inactivated, the engineered overproduction of the M13 gene X product mediates phage replication repression. M13-102 contains two new additions: (1) a sequence enabling triple-helix-mediated affinity capture (TAC) for purification of linearized vector DNA, and (2) universal primer sequences for wider compatibility with commercial instruments that support fluorescence-based sequencing. Using a biotinylated homopyrimidine oligodeoxyribonucleotide as third-strand probe, TAC is performed on streptavidin-coated magnetic beads [Ji et al., Genetics Analysis: Techniques and Applications 11 (1994) 43-47], and serves as a rapid and efficient alternative to gel purification. To reduce tandem insertions, phosphatase treatment of insert DNA was easily invoked without sacrificing cloning efficiency. The combined capabilities of direct selection, TAC purification and phosphatase treatment of inserts should facilitate library construction and improve overall library quality.

The genome-scale data collected by the FANTOM4 collaborative research project are presented as an integrated web resource., In FANTOM4, an international collaborative research project, we collected a wide range of genome-scale data, including 24 million mRNA 5'-reads (CAGE tags) and microarray expression profiles along a differentiation time course of the human THP-1 cell line and under 52 systematic siRNA perturbations. In addition, data regarding chromatin status derived from ChIP-chip to elucidate the transcriptional regulatory interactions are included. Here we present these data to the research community as an integrated web resource.