We described in a previous post Ensembl’s new regulatory annotation of the genome. Now, we will go in greater detail into how we computed it.

We started by running ChromHMM over 17 cell types, using publicly available ENCODE and Roadmap Epigenomics data. This produced a segmentation or annotation the genome for each of these cell types under 25 labels, or segmentation states. These states were given arbitrary names (P0, P1, …) after a preliminary comparison to the earlier ENCODE segmentations across six cell types.

For each state and each position in the genome, we computed the number of cell types that have that state at that position. This resulted in 25 segmentation state summary tracks. We also pulled in all the Chip-Seq peaks from the January 2011 ENCODE data freeze, and kept those that overlapped with open chromatin (i.e. DNAseI hypersensitivity) on the same cell type. From all these assays, we computed a summary track, which indicates the probability (between 0 and 1) of seeing a TF peak at any location of the genome. The segmentations and summary tracks can be seen in the illustration below.

Summary tracks generated from the segmentation and TFBS peaks

We then used TF binding data to determine the specificity of the ChromHMM states, as regulatory regions are presumably correlated with TF binding. Each function (TSS, CTCF insulator, proximal regulatory elements, distal regulatory elements) was thus associated with several states. For example, transcription start sites were strongly associated to the P0 state in the segmentations. We overlapped these state signals to define function specific signals. A simple count threshold was set to maximise the detection of TF binding sites. This led to the regions as displayed at the bottom of the following figure:

Selected summary tracks were overlapped to define the Ensembl Regulatory features

These new regions concur strongly with the TF binding datasets: 73.4% of the TF Chip-Seq peaks were captured in the ChromHMM regions, equivalent to an 8.3x enrichment with respect to the genomic average. Conversely, 24.0% of the ChromHMM-based regions were covered by observed TF Chip-Seq peaks. To avoid losing information, TFBS peaks which were not covered by any of these elements were marked as ‘Unannottated TFBS’.

Having defined consensus regulatory region, we returned to the original data, to determine which region is active in which cell line:

The Regulatory Features are then compared to the cell type specific segmentations to determine their activity in each cell type.

Validation

• Fantom 4 CAGE tags

In the median cell type, 83% of FANTOM tags supported by three CAGE tags or more were annotated by our pipeline.

• Vista enhancers

The Vista Enhancer database contains enhancer sequences validated by in vivo staining assays on transgenic mice (hats off to the VISTA team for their years of meticulous work). It currently contains 1,575 predicted enhancers, of which 807 were experimentally confirmed. 491 of those (60.8%) were picked up by our pipeline.

• TF binding motifs

We found two estimates of active TF binding motifs:

JASPAR motifs are not mapped by default to the human genome, so we are missing a few. We will therefore be remapping them in time for Ensembl release 75, in early 2014.

The computing of Ensembl’s new regulatory annotation is work in progress. If you have any ideas on the subject, feel free to leave a comment or to send them to helpdesk@ensembl.org.

GWAS after GWAS return statistically significant hits that are hard to interpret because they fall outside of coding regions, and this begs for more functional annotation of regulatory regions. We at Ensembl have been providing such an annotation for a few years now and we are now redesigning from the ground up the way we define these regions. This work is in progress and we would love to hear your suggestions and comments.

An overview of the major elements involved in gene expression regulation

In short, we are looking for all regions of the genome which display regulatory function. Much ink has been spilled over the definition of the word ‘functional’, so we’re going to expand a bit.

We propose to map out the regions of the genome that display epigenomic marks and/or transcription factor binding sites (TFBS) associated to proximal and distal regulatory elements, transcription start sites (TSS), and CTCF insulators.

Ensembl’s Regulation pipeline

We will post more details next week on Computing the New Ensembl Regulatory Annotation. To cut to the chase, we defined the following regions from publicly available ENCODE and Roadmap Epigenomics datasets:

The new Regulatory Build will allow us to separate state from function, as shown below, upstream of VNN2:

The track at the top colours the region by function, independently of the cell type. In the cell specific tracks below, the various features are greyed out if we do not have evidence of activity for that region.

We incorporated our preliminary results into a track hub, along with some of our intermediary data. Next week we will post more details on Computing Ensembl’s New Regulatory Build. We want to integrate this build officially into Ensembl release 76, sometime during the 3rd or 4th quarter of 2014.

FAQ:

Are you saying that 9.7% of the genome is functional?

Not quite. We’re saying that if you split the genome into 200 bp bins, 9.7% of them show epigenomic marks or TF binding. Remember that histone marks are measured at nucleosome resolution, so this signal is at best at a 140bp resolution. If you add in experimental noise (typically proportional to the Chip-Seq fragment length), the exact position of these elements on the genome is rather fuzzy. At the same time, the epigenome is a dynamic system, and we only have some assays on some cell lines. No doubt more regions will be annotated as more datasets come in.

What happened to the 80.4% of the genome being functional?

This statistic from the main ENCODE paper took into account other biochemical markers, in particular those associated to transcription, which can be observed over most of the genome. We therefore recommend using curated genesets such as GENCODE to define gene bodies. Nonetheless, the number of regulatory elements and promoters described here is of the same order of magnitude as that discussed in the ENCODE paper.

Is this the same as ENCODE?

This is more than just ENCODE. There are other fascinating epigenomic surveys out there, such as Roadmap Epigenomics or BLUEPRINT to name a few. Here at Ensembl, we have started merging all these datasets (including ENCODE), and provide the most comprehensive overview possible, updating our calls as new projects come along. Also, as discussed above, we are producing a cell-type independent summary of epigenomic function, which can be used to inform studies on new cell types.

What about other species?

We focused our Regulation database primarily on human: that is what most of our users ask for, and what we have most data for. But that does not mean that we ignore other species. Ensembl has already regulatory information for mouse, and we plan on shortly expanding this to farm animals, in collaboration with the Roslin Institute.

Can you assign regulatory elements to genes?

We’re working on it. Correlations are easy to find, but multiple testing quickly gets in the way when testing 310,287 regulatory elements against 40,249 TSSs.

Remember, this is work in progress, and we would love to hear your suggestions. Please leave your comments here or drop us a line.

Today I am happy to announce Ensembl’s migration away from CVS as our primary version control system (VCS) to Git. This migration sees the end of nearly a year’s worth of work to ensure that our Git repositories provide the same historical record as CVS.

To summarise the changes:

• Ensembl’s code is now provided from our GitHub organisation at http://github.com/Ensembl
• We have migrated Ensembl’s versioning back to 1999
• We will continue to back-port changes to CVS for the next 3 releases (support ending with release 77)
• You can still download our API release tarballs from our FTP site

CVS (Concurrent Version System) was first released in 1990 and was based on an earlier system called RCS (released in 1982). It relies on a centralised single server to hold all previous revisions with none of this information held on the client. CVS also assumes that commits to files within the same project are independent of each other. When the Ensembl project started in 1999, we chose CVS as it was one of the best available VCS in the open source community.

Since that choice, a new breed of VCS has appeared: decentralised/distributed version control systems (DVCS). These systems favour local copies of the repositories, removing the need to communicate with a centralised server, except when sending or receiving new commits, and work with sets of file changes as a single atomic block of work. According to Black Duck’s comparison of repositories, Git is the dominant DVCS in open source projects. We have decided to use the code hosting company GitHub as the location of our repositories. GitHub has been a major contributor behind the success of Git by providing an infrastructure that promotes social coding between developers.

Whilst CVS has been a very good servant to Ensembl, the time has come to move on to better tools. We have seen other projects within EMBL-EBI and the Wellcome Trust Sanger Institute make a similar transition to Git. None of them have looked back, citing better tooling, a larger support base and an ability to support both long-term and short-term development branches. We agree and cannot wait to start using this exciting technology.

Monday morning in London: hustle and bustle of the start of the working week.
Packed trains and tube. Huge crowds on their customary way to work.

Congestion on the London underground.

I too (armed with my laptop, a course booklet and a pen) was on my way to London to deliver training in Ensembl.

Aida Santaolalla and Anita Grigoriadis organised this Ensembl browser workshop for the cancer research community at Guy’s hospital, a large NHS hospital with breathtaking views of the River Thames.

View of London from Guy’s hospital.

We had 18 participants with mixed professional roles within the hospital: postdocs, PhD students, staff researchers, principal investigators, and a physician. All had one research theme in mind: cancer.

Cancer is a broad range of diseases characterised by unregulated cell growth due to varied and complex causes, one of them being genetic predisposition. During the workshop, I demonstrated, among other things, how Ensembl could be used to find out genes and variants (i.e. SNPs, CNVs and indels) that may confer such genetic predisposition. The location of variants that are associated with breast cancer, for instance, can be seen in Ensembl.

Among the attendees, 40% were completely new to Ensembl and a whopping 80% had never used Ensembl BioMart before. The comments received in my feedback survey were very rewarding: ‘This was a very good workshop where I learned the basics of Ensembl software’, ‘This workshop was particularly useful in pointing out tools in Ensembl that I wouldn’t have known existed through personal investigation.’ And better still, every single participant indicated they would use Ensembl and Ensembl BioMart more often after this workshop, and they would recommend the workshop to a colleague.

Ensembl workshop delivered. Happy participants. Great feedback. Job done.
The day was far from over though. I still had to make my way out of the city.

Monday evening in London: hustle and bustle of the end of the first day of the working week. Packed trains and tube: huge crowds on their customary way home.

Can’t deny I was chuffed to bits to go back to the HQs of Ensembl in the quiet, tranquil Cambridgeshire.

Now I’m ready for my next adventure: University of Pavia, Italy.
Maybe I will come to your institute one day.

If you want to organise a training session in Ensembl, you can follow the steps of our hostesses at Guy’s, who contacted us to organise this successful Ensembl workshop in London for the cancer research community.

Today the Wellcome Trust Sanger Institute and the European Bioinformatics Institute (EMBL-EBI) announced plans to reorganise the Ensembl project so that we can best leverage the strengths of Ensembl’s parent institutes to capitalise on emerging opportunities in genomics

From our users’ perspective, all existing Ensembl services including our genome browser, APIs and regular releases will continue as usual. Behind the scenes, the current Ensembl services will be consolidated at EMBL-EBI to help us strengthen the existing resources and facilitate closer links with UniProt, Ensembl Genomes and the Expression Atlas, which are all based at EMBL-EBI. Ensembl will also continue to be closely involved in the GENCODE project coordinated at Sanger.

New Ensembl activities that focus on novel methods for storing and representing human variation will be based at the Sanger Institute. These efforts will be aligned with the aims of the global alliance for the secure sharing of genomic and clinical data. As part of the reorganisation, Ensembl will also be connected more closely with the DECIPHER project. DECIPHER is an interactive, web-based service to support sharing of likely functional, rare clinical variation and engagement with the clinical community.  These connections will improve clinically relevant access to the rich genome annotations provided within Ensembl.

This is an extraordinary time for genomics. Ensembl has supported and contributed to the dramatic advances in genomics over almost 15 years and we are excited about what the future holds.

Please note that the archive websites for Ensembl release 60 (Nov 2010) will be retired in mid December when version 74 is released.

This is in accordance with our rolling retirement policy, whereby archives more than three years old are retired unless they include the last instance of the previous assembly from one of our key species (human, mouse and zebrafish).

For more information about how to use archives, please see our previous blog post on the topic; a list of all current archives is available on the main website.

The Genome Reference Consortium (GRC) plans to release a new human assembly (GRCh38) later this year. What is the reason for an update? The current human genome in Ensembl, UCSC and NCBI (GRCh37) is indeed high quality. The GRC reports it’s accurate to an error rate of ~1 in 100,000 bases. However, there are still gaps in the assembly and there are a number of difficult loci still to be resolved.  The new update addresses many of these issues.  More reasons for the update can be found on the GRC blog.

The new assembly will be available in Ensembl next year (the third or fourth quarter of 2014). What happens between the public release of the updated human assembly and the Ensembl release of the annotated genome? A series of posts from our team will cover the work required to annotate genes, variants, and more to a high standard. These articles will reflect not only the efforts to deliver high quality annotation, but to integrate the data in a useful way for our users. At least one post each month will reflect our thorough analysis of the genome in the following areas:

• Release cycle (how does it work?)
• The GRCh38 assembly
• The new regulation build– integration of ENCODE and Blueprint data
• Coordinate mapping from one assembly version to another
• Processing dbSNP and other sources of variants
• High quality genes, annotating the GENCODE set
• Updates to the Variant Effect Predictor (VEP)
• Determining stable ids for the new gene set
• Whole Genome Alignments– pairing up the new human assembly with other species
• Quality control- how do we check our data?

Keep your eye on our blog for more posts in this series, marked with the category “GRCh38 Ensembl”.

Are the Ensembl databases and API a core part of your research? Did you develop tools and scripts to get exactly what you need from these resources? Why not share them with the community?

Ensembl are pleased to announce e!code, a directory of programming resources for use with the Ensembl datasets and codebase. It currently includes a selection of VEP plugins developed by the Ensembl team plus external contributions such as the Java API JEnsembl.

Please note that this new site is not a repository, only a central listing of available resources. If you would like to contribute, please be sure to read our contributor guidelines.

Visit our e!code mini-site (part of this blog) for more information!

New Pre! sites have been released for four species: the Mexican blind cave fish (Astyanax mexicanus), southern white rhinoceros (Ceratotherium simum simum), prairie vole (Microtus ochrogaster) and armadillo (Dasypus novemcinctus).

The prairie vole assembly, MicOch1.0 (GCA_000317375.1), was made available by The Broad Institute in October 2012. The prairie vole is a model for social behaviour. The animals live in colonies and have been known to display aspects of human-like behaviour such as lifelong pair bonding. The vole assembly is composed of 21 chromosomes and 6314 unplaced scaffolds. Click here to go to the vole Pre! site, where you can view alignments of vole proteins from Uniprot, and mouse and human Ensembl translations.

The armadillo assembly, Dasnov3.0 (GCA_000208655.2), was made available by the Baylor College of Medicine in December 2011. The armadillo is a natural reservoir of leprosy that can be acquired by humans who handle or consume them. The species is also used to study multiple births and delayed implantation of embryos, they usually produce identical quadruplets. The armadillo assembly is composed of 46558 unplaced scaffolds. Click here to go to the armadillo Pre! site, where you can view alignments of the armadillo Uniprot proteins and human Ensembl translations.

The cave fish assembly, AstMex102 (GCA_000372685.1), became available in April 2013. The species has both surface dwelling (surface fish) and cave adapted (cave fish) morphs and is an important model in evolutionary biology. The cavefish differ from the surface fish in several traits such as the enhancement of non-visual sensory systems and the loss of eyes and pigmentation. As the two different morphs are inter-fertile the species is a useful model for microevolution studies, although it is primarily used as a model for retinal degeneration diseases. The cave fish assembly comprises 10735 unplaced scaffolds. Click here to go to the cavefish Pre! site, where you can view alignments of cavefish Uniprot proteins, and stickleback and zebrafish Ensembl translations.

The southern white rhinoceros assembly, CerSimSim1.0 (GCA_000283155.1), was made available by the Broad Institute in August 2012.  This particular species is a valuable model for a number of reasons. Potential longevity has been estimated to be between 40 and 50 years, and although they may be sexually mature by the age of 4, often they do not reproduce until much later in life. They are also important for comparative studies with horses, having diverged about 55mya. Finally, rhino studies are important for conservation reasons as they are near threatened species due to poaching. The rhino assembly comprises 3086 unplaced scaffolds. Click here to go to the rhinoceros Pre! site, where you can view alignments of the rhinoceros Uniprot proteins and human Ensembl translations.

I’ve been working for EMBL, the European Molecular Biology Laboratory, for around nine months now, and only last week did I really start to understand the organisation that I’m part of. Like two-thirds of the Ensembl team, I work at the European Bioinformatics Institute on the Genome Campus near Cambridge, a campus we share with the Wellcome Trust Sanger Institute, which hosts the other third of Ensembl.

Last Friday I attended EMBL Lab Day, a conference/celebration for scientists working at all of the five EMBL sites around Europe, held in EMBL’s fabulous helix-shaped ATC in Heidelberg. I was looking forward to speaking to the EMBL audience about Ensembl and all the exciting new data and tools we’ve been adding recently (see other entries in this blog to see our recent news). In return, I was excited to find out about all the interesting lab work and data that came out of EMBL, and to get talking to some of my EMBL colleagues.

I was not disappointed. There was such a variety of interesting work being presented in the talks, from new methods of identifying protein structures, to studying the development of haematopoietic cells and the factors affecting cancer recurrence. I was fascinated to learn about new methods to manipulate cell activity using light pulses and how platelets, such small and seemingly simple cells, are able to reorganise their structure on activation using their cytoskeleton. The talks were all short, 12 minutes plus questions, giving enough time to get a taster of the work without getting bogged down with details. Most of the presenters were at a relatively early stage of their research careers, making Lab Day an important opportunity for the speakers to get experience speaking to a receptive and varied audience.

The EMBL choir singing at Lab Day – courtesy of Adam Gristwood, EMBL

There were long breaks in between the talk sessions, so we had plenty of time to network with other EMBLers and look at the posters, which were displayed all the way up each side of the helix, both serious posters detailing the work of the labs, and fun posters introducing the lab members. Congratulations to Matthias Ziehm from Janet Thornton’s group at the EBI who won a poster prize.

Once the talks were over we celebrated the achievements of young EMBL scientists with a PreDoc (that’s EMBL-speak for PhD student) graduation ceremony, followed by a champagne reception. Afterwards, there were performances by the EMBL choir and drum group, then we had a disco. I made lots of new friends and even got invited to a barbeque on the banks of the Necker in Heidelberg the following evening.

Coming back from Heidelberg, I was struck by two things about EMBL. Firstly, EMBL is a hub of scientific excellence, producing fantastic science and training up young scientists both in and out of the lab to become the scientific leaders of the future. Secondly, it’s just such a nice place to work, promoting scientific and social interaction between its members in so many ways and celebrating the achievements of its members. I’m glad to be part of the EMBL family, and I’m looking forward to Lab Day next year.

If you think you’d like to join EMBL, take a look at the EMBL jobs page to find out more about jobs and fellowships.