Jessica Severin

EnsemblCompara GeneTrees: Complete, duplication-aware phylogenetic trees in vertebrates

We have developed a comprehensive gene orientated phylogenetic resource, EnsemblCompara GeneTrees, based on a computational pipeline to handle clustering, multiple alignment, and tree generation, including the handling of large gene families. We developed two novel non-sequence-based metrics of gene tree correctness and benchmarked a number of tree methods. The TreeBeST method from TreeFam shows the best performance in our hands. We also compared this phylogenetic approach to clustering approaches for ortholog prediction, showing a large increase in coverage using the phylogenetic approach. All data are made available in a number of formats and will be kept up to date with the Ensembl project.

Gateways to the FANTOM5 promoter level mammalian expression atlas

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.

An atlas of human long non-coding RNAs with accurate 5′ ends

Long non-coding RNAs (lncRNAs) are largely heterogeneous and functionally uncharacterized. Here, using FANTOM5 cap analysis of gene expression (CAGE) data, we integrate multiple transcript collections to generate a comprehensive atlas of 27,919 human lncRNA genes with high-confidence 5′ ends and expression profiles across 1,829 samples from the major human primary cell types and tissues. Genomic and epigenomic classification of these lncRNAs reveals that most intergenic lncRNAs originate from enhancers rather than from promoters. Incorporating genetic and expression data, we show that lncRNAs overlapping trait-associated single nucleotide polymorphisms are specifically expressed in cell types relevant to the traits, implicating these lncRNAs in multiple diseases. We further demonstrate that lncRNAs overlapping expression quantitative trait loci (eQTL)-associated single nucleotide polymorphisms of messenger RNAs are co-expressed with the corresponding messenger RNAs, suggesting their potential roles in transcriptional regulation. Combining these findings with conservation data, we identify 19,175 potentially functional lncRNAs in the human genome.

Interactive visualization and analysis of large-scale sequencing datasets using ZENBU

217 clustering (Fig. 1c and Supplementary Figs. 3 and 4), and collation (Fig. 1h and Supplementary Fig. 3). With an understanding of these atomic operations, more advanced users can combine them into complex processing scripts. These customized scripts can also be saved and shared allowing efficient re-use of optimized analyses and views. To demonstrate some of ZENBU’s functionality, we show multiple views on the same underlying data, ENCODE RNA-seq2 experiments loaded in BAM format (Fig. 1). One of the more powerful views in this figure is the dynamic projection of RNA-seq reads onto GENCODE11 transcript models to calculate RPKM (reads per kilobase transcript per million reads) values (Fig. 1h). Transcripts are then colored by support and the RPKM expression table for these models can be downloaded in a variety of formats. A variation of the same script projects CAGE expression signal into the proximal promoter regions (±500 bp of 5ʹ end) of the same models (Supplementary Fig. 2). Furthermore, an implementation of the parametric clustering approach paraclu12 allows ZENBU to identify peaks of CAGE signal (Fig. 1c) and extract their corresponding expression values per experiment. Paraclu imposes minimal prior assumptions and is flexible enough for use with small RNA and ChIP-seq data by varying the clustering parameters (Supplementary Fig. 3 and 4). ZENBU also allows investigators to fine tune these settings and rapidly inspect the results on specific loci before genome-wide computation. A more detailed case study recapitulating an integrated ChIP-seq and RNA-seq analysis13 is included in Supplementary Note 1 to demonstrate the power of the complex scripting possible in ZENBU. To enable fast and reliable downloading of data genome-wide and to speed up visualization and processing, we implemented a track caching system, which To the Editor: The advance of sequencing technology has spurred an ever-growing body of sequence tag–based data from protocols such as RNA-seq, chromatin immunoprecipitation (ChIP)-seq, DNaseI-hypersensitive site sequencing (DHS)-seq and cap analysis of gene expression (CAGE). Large-scale consortia are applying standardized versions of these protocols across broad collections of samples1,2 to elucidate genomic function. Beyond this, the affordability of these systems and availability of sequencing services has made these technologies accessible to smaller laboratories focusing on individual biological systems. Data generation is only the beginning, however, and a substantial bottleneck for many labs is going from sequence data to biological insight, especially when the volume of data overwhelms standard paradigms for data visualization. Here, we present a web-based system, ZENBU, which addresses this problem by extending the functionality of the genome browser (Supplementary Fig. 1). For users with limited bioinformatics skills, ZENBU provides a suite of predefined views and data-processing scripts optimized for RNA-seq (Fig. 1), CAGE, short-RNA and ChIP-seq experiments (Supplementary Figs. 2–4). These are available simply upon uploading data as BAM (binary version of the sequence alignment/map)3 files. The system can also generate an optimized set of views for each of the above data types. ZENBU provides a rich selection of data manipulation capabilities, including quality filtering, signal thresholding, signal normalization, peak finding, annotation, collation of signal under peaks or transcript models and expression difference visualization across multiple experiments. We designed ZENBU with large-scale transcriptome projects in mind. In particular, the FANTOM5 project (unpublished data) required a system that would allow rapid incremental data loading, visualization, interpretation and downloading of thousands of deep CAGE, RNA-seq and small-RNA data sets as they were produced. We reviewed available systems4, including genome browsers, such as University of Tokyo Genome Browser (UTGB)5, Gbrowse6, University of Santa Cruz (UCSC) genome browser7, Ensembl8 and Integrative Genome Viewer (IGV)9, and data management tools, such as Biomart10, and found none with the full set of functions that we sought (see Supplementary Table 1 for comparison). Therefore, we developed ZENBU, a stable, fast, efficient and secure system that is flexible enough to allow customization of data filters, views and analyses. A key feature of ZENBU is that it allows the user to combine multiple experiments on demand (up to thousands of experiments) within any single track and interpret the data through linked views (Fig. 1). Upon combining multiple experiments, the genome browser view shows the combined genomic distribution of tags from these experiments within a given genomic interval (Fig. 1d), conversely the linked expression view shows the relative abundance of tags observed in this region across these experiments (shown as a histogram, Fig. 1j). The genome browser and expression view are ‘linked’, meaning that as the user interacts with one view the other is updated in real-time. This facilitates interactive exploration of the data because selecting features or regions within a browser track results in data for only that region being displayed in the expression view. And symmetrically, hiding specific experiments in the expression view updates the data displayed in the browser track. Data processing and complex views are achieved by a flexible on-demand scripting system based on data transformation and analysis modules. A selection of predefined combinations of simple operations are provided to perform generic tasks such as data normalization (Fig. 1b–d,h,i), data filtering (Supplementary Fig. 2), data Interactive visualization and analysis of large-scale sequencing datasets using ZENBU correspondence

Single-Molecule Approach to Bacterial Genomic Comparisons via Optical Mapping

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 of Shigella flexneri serotype 2a (2457T and 301), Yersinia pestis (CO 92 and KIM), and Escherichia coli were aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequenced Shigella flexneri strain (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.

A Whole-Genome Shotgun Optical Map of Yersinia pestis Strain KIM

ABSTRACT 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 (XhoI and PvuII) 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 XhoI map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the PvuII 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.

Behavioral and social correlates of escape from suppression of ovulation in female common marmosets housed with the natal family.

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. Am J Primatol 41:1–21, 1997.

Promoter architecture of mouse olfactory receptor genes

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 gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.

Update of the FANTOM web resource: high resolution transcriptome of diverse cell types in mammals

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.

Technical Advance: Transcription factor, promoter, and enhancer utilization in human myeloid cells

The generation of myeloid cells from their progenitors is regulated at the level of transcription by combinatorial control of key transcription factors influencing cell‐fate choice. To unravel the global dynamics of this process at the transcript level, we generated transcription profiles for 91 human cell types of myeloid origin by use of CAGE profiling. The CAGE sequencing of these samples has allowed us to investigate diverse aspects of transcription control during myelopoiesis, such as identification of novel transcription factors, miRNAs, and noncoding RNAs specific to the myeloid lineage. We further reconstructed a transcription regulatory network by clustering coexpressed transcripts and associating them with enriched cis‐regulatory motifs. With the use of the bidirectional expression as a proxy for enhancers, we predicted over 2000 novel enhancers, including an enhancer 38 kb downstream of IRF8 and an intronic enhancer in the KIT gene locus. Finally, we highlighted relevance of these data to dissect transcription dynamics during progressive maturation of granulocyte precursors. A multifaceted analysis of the myeloid transcriptome is made available (www.myeloidome.roslin.ed.ac.uk). This high‐quality dataset provides a powerful resource to study transcriptional regulation during myelopoiesis and to infer the likely functions of unannotated genes in human innate immunity.