Demo: Introduction to Ensembl

Ensembl

Homepage

The front page of Ensembl is found at ensembl.org. It contains lots of information and links to help you navigate Ensembl:

On the right-hand panel you can see the current release number and what has come out in this release. To access old releases, scroll to the bottom of the page and click on View in archive site in the right-hand corner.

Click on the links to go to the archives. Alternatively, you can jump quickly to the correct release by adding e plus the release number in the URL. For example e98.ensembl.org jumps to Ensembl release 98.  
 
 

Available species

Scroll back up to the top of the homepage. You can view all available species by clicking the View full list of all species link underneath the coloured search block.

You can search for your species of interest (either the common or scientific name) using the search bar at the top right-hand corner of the table. Click on the common name of your species of interest to go to the species information page. We’ll click on Human.

Species information

Here you can see links to example features and to download flatfiles. To find out more about the genome assembly and genebuild, click on More information and statistics under the Genome assembly section.

Here you’ll find a detailed description of how to the genome was produced and links to the original source. You will also see details of how the genes were annotated.

The current genome assembly for human is GRCh38. If you want to see the previous assembly, GRCh37, visit our dedicated site grch37.ensembl.org.

Ensembl Genomes

Homepage

Let’s take a look at the Ensembl Genomes homepage at ensemblgenomes.org.

Click on the different taxa to see their homepages. Each one has a different colour-coding, but they are all structured in a similar format to the Ensembl main site.

You can navigate most of the taxa in the same way as you would with Ensembl.  
 
 

Ensembl Bacteria

Ensembl Bacteria has a large number of genomes and has a slightly different method to the other Ensembl sites. Let’s look at it in more detail.

There’s no drop-down species list for bacteria as it would be hard to navigate with the number of species. You can click the View full list of all Ensembl Bacteria species link underneath the coloured search block. Search for your species of interest using the filter in the top right-hand corner of the table.

Alternatively, you can find a species by typing the species name into the Search for a genome search box at the top of the page. A drop-down list will appear with any species matching the name you entered.

For example, to find a sub-strain of Clostridioides difficile start typing in the species name. Due to the auto-complete, you’ll see useful results as soon as you get to Clostridio.

The drop down contains various strains of C. difficile. Let’s choose C. difficile 630. This will take us to another species information page, where we can explore various features.

Unlike the Homo sapiens species information page, there is no prose description of the genome or gene annotation, as these pages were generated automatically.  
 
 

Ensembl Rapid Release

Our newest genomes, such as those coming from the Darwin Tree of Life, are available rapid.ensembl.org with limited annotation.

Mycobacterium tuberculosis H37Ra str. ATCC25177 is a clinical strain.

Go to Ensembl Bacteria and find the species M. tuberculosis H37Ra str. ATCC25177. How many coding genes does it have?

Go to Ensembl Fungi and answer the following questions:

1. How many genomes of the genus Fusarium are in Ensembl Fungi?

2. What is the accession ID of the Fusarium graminearum str. CS3005 INSDC assembly?

3. How many genes have been annotated for F. graminearum str. CS3005?

Find the species Oryza sativa Japonica in Ensembl Plants. How many coding and non-coding genes does it have?

Go to Ensembl Plants and answer the following questions:

1. How many genomes of the genus Solanum are there in Ensembl Plants?

2. When was the current Solanum lycopersicum genome assembly last revised?

Go to Ensembl and find the following information:

1. What is the name of the genome assembly for Panda?

2. How long is the Panda genome (in bp)? How many coding genes have been annotated?

Region in Detail view

Start at the Ensembl Bacteria homepage, bacteria.ensembl.org. Search for your species of interest either by using the search box, or opening the full list of species by clicking View full list of all Ensembl Bacteria species underneath the search box.

Enter Escherichia coli str. K-12 substr. MG1655 (GCA_000005845) in the search box. Enter Chromosome:3144663-3157453 into the species-specific search box:

Press Enter or click Go to jump directly to the Region in detail page.

Click on the button to view page-specific help. The help pages provide text, labelled images and, in some cases, help videos to describe what you can see on the page and how to interact with it.

The Region in detail page is made up of three images, let’s look at each one on detail.

The first image shows the chromosome:

You can jump to a different region by clicking and dragging the yellow and blue handles.

If you want to move to your highlighted region, you click on the region shaded in red.

The second image shows a 50 kb region (the size varies per genome and depends on the gene size and density; you can find a scale at the top of the view) around our selected region. This view allows you to scroll back and forth along the chromosome.

Click and drag your mouse to highlight a region. A pop-up window will appear with options to jump to or centre on the highlighted region.

Click on the X to close the pop-up menu.

Click on the Drag/Select button to change the action of your mouse click. Now you can scroll along the chromosome by clicking and dragging within the image. As you do this you’ll see the image below grey out and update to your scrolled region. To go back to go back to where you started, you can click the Back button of your browser.

The third image is a detailed, configurable view of the region.

Click on the Drag/Select option at the top or bottom right to switch mouse action. On Drag, you can click and drag left or right to move along the genome, the page will reload when you drop the mouse button. On Select you can drag out a box to highlight or zoom in on a region of interest.

We can edit what we see on this page by clicking on the blue Configure this page menu at the left.

This will open a menu that allows you to change the image.

You can enable tracks in different styles; more details are in the FAQs.

Let’s add the following tracks to our view:

• Start/stop codons
• All repeats

Now click on the check icon in the top left-hand corner to save and close the menu. Alternatively, click anywhere outside of the menu. We can now see the tracks in the image.

We can also change the way the tracks appear by clicking on the track name then hovering over the cog wheel to open its menu. We can move tracks around by clicking and dragging on the bar to the left of the track name.

Now that you’ve got the view how you want it, you might like to show something you’ve found to a colleague or collaborator. Click on the Share this page button to generate a URL with your set configurations. Email the link to someone else, so that they can see the same view as you, including all the tracks you’ve added. These links contain the Ensembl release number, so if a new release or even assembly comes out, your link will just take you to the archive site for the release it was made on.

To return this to the default view, go to Configure this page and select Reset configuration at the bottom of the menu.

Go to the Ensembl Bacteria homepage and do the following:

1. Search for the Staphylococcus aureus subsp. aureus NCTC 8325 (GCA_000013425).

2. Search for the gene gyrA.

3. What are the genomic coordinates of this gene? Is gyrA located on the forward or reverse strand?

4. Name two genes located upstream and downstream of gyrA.

Go to Ensembl Bacteria and do the following:

1. Search for the Salmonella enterica subsp. enterica serovar Typhi str. Ty2 (GCA_000007545) (Hint: type Ty into the Search for a genome box).

2. Go to the region Chromosome:2000605-2009742.

3. How many genes are annotated in this region? How many are on the forward strand? How many are on the reverse strand?

Go to Ensembl Fungi. Let’s try to find some information about the region from 1,400,000 to 1,425,000 in chromosome 7 in Coprinopsis cinerea okayama:

1. How many complete genes are found in this region? How many on the forward and how many on the reverse strand?

2. Zoom in on the largest gene EFI27358. How many exons does this gene have?

3. Export the genomic sequence in FASTA format for this region.

Go to the Ensembl Plants homepage and do the following:

1. Go to the region 1:405000-453000 in Oryza sativa Japonica.

2. Turn on the AGILENT:G2519F-015241 microarray track. Are there any oligo probes that map to this region?

3. Highlight the region around any reverse strand probes you can see. Do they map to any transcripts?

Go to Ensembl.

1. Go to the region from 32,264,000 to 32,492,000 bp on human chromosome 13. On which cytogenetic band is this region located? How many contigs make up this portion of the assembly (contigs are contiguous stretches of DNA sequence that have been assembled solely based on direct sequencing information)?

2. Zoom in on the BRCA2 gene.

3. Configure this page to turn on the LTR (repeat) track in this view. What tool was used to annotate the LTRs according to the track information? How many LTRs can you see within the BRCA2 gene? Do any overlap exons?

4. Create a Share link for this display. Email it to your neighbour. Open the link they sent you and compare. If there are differences, can you work out why?

5. Export the genomic sequence of the region you are looking at in FASTA format.

6. Turn off all tracks you added to the Region in detail page.

Go to the Ensembl homepage.

1. Go to the region from 150,320,000 to 150,540,000 bp on mouse chromosome 5. How many contigs make up this portion of the assembly (contigs are contiguous stretches of DNA sequence that have been assembled solely based on direct sequencing information)?

2. Zoom in on the Brca2 gene.

3. Configure this page to turn on the LTR (repeat) track in this view. What tool was used to annotate the LTRs according to the track information? How many LTRs can you see within the Brca2 gene? Do any overlap exons?

4. Create a Share link for this display. Email it to your neighbour. Open the link they sent you and compare. If there are differences, can you work out why?

5. Export the genomic sequence of the region you are looking at in FASTA format.

6. Turn off all tracks you added to the Region in detail page.

Demo: Viewing genes and transcripts

You can find out lots of information about Ensembl genes and transcripts using the browser. If you’re already looking at a Region in detail view, you can click on any transcript and a pop-up menu will appear, allowing you to jump directly to that gene or transcript.

Alternatively, you can find a gene by searching for it. You can search for gene names, identifiers, or functions that might be associated with the genes.

We’re going to look at the lacZ gene Escherichia coli str. K-12 substr. MG1655 (GCA_000005845). From bacteria.ensembl.org, search for the Escherichia coli_ str. K-12 substr. MG1655 (GCA_000005845) genome. Type lacZ into the species-specific search bar and click the Go button.  
 
 

The gene tab

Click on the gene ID b0344 from the search hits. The Gene tab should open:

This page summarises the gene, including its location, name and equivalents in other databases. At the bottom of the page, a graphic shows a Region in detail view with the transcripts. We can also see the overlapping and neighbouring genes.

There are different tabs for different types of features, such as genes and transcripts. These appear side-by-side underneath the species name at the top of the page, allowing you to jump back and forth between features of interest. Each tab has its own navigation column down the left hand-side of the page, listing all the things you can see for this feature.

Gene sequence

Let’s walk through the menu for the Gene tab. Click Sequence in the left-hand panel to view the genomic sequence of the gene.

The sequence is shown in FASTA format. The FASTA header contains the genome assembly, chromosome, coordinates and strand (1 or -1). This gene is on the positive strand.

Exons are highlighted within the genomic sequence: the exon of our gene of interest and any neighbouring or overlapping genes. By default, 600 bases are shown up and downstream of the gene. We can make changes to how this sequence appears with the Configure this page button found at the left. This allows us to change the flanking regions, add line numbering and more. Click on it now.

We have changed our Flanking sequences to 200 and added Line numbering relative to the coordinate system. Save your setting by clicking the check icon at the top right-hand corner.

You can download this sequence by clicking in the Download sequence button above the sequence. This will open a dialogue box that allows you to pick between plain FASTA sequence, or sequence in rich-text format (RTF), which includes all the coloured annotations and can be opened in a word processor. If you want run a sequence analysis tool, download as FASTA sequence, whereas if you want to analyse the sequence visually, RTF is best for this. This button is available for all sequence views.

Gene function

To find out the protein function, have a look at gene ontology (GO) terms from the Gene Ontology consortium. There are three pages of GO terms, representing the three divisions: GO: Biological process (what the protein does)
GO: Cellular component (where the protein is)
GO: Molecular function (how it does it)

Click on GO: Biological process to see an example of the GO pages.

Here you can see the functions that have been associated with the gene. There are three-letter codes that indicate how the association was made, as well as links to the specific transcript they are linked to.  
 
 

Gene information in external databases

We also have links out to other databases which have information about our genes and may focus on other topics that we don’t cover, like the European Nucleotide Archive ENA) or the UniProt knowledge base UniProtKB. Go up the left-hand menu to External references:

The transcript tab

We’re now going to explore the transcript of lacZ. Click on Show transcript table underneath the gene summary at the top of the page.

Here we can see a list of all the transcripts of lacZ with their identifiers, lengths and biotypes. The lacZ gene only has one transcript. Click on the transcript ID AAC73447.

You are now in the Transcript tab for AAC73447. We can still see the gene tab so we can easily jump back. The left hand navigation column provides several options for the transcript AAC73447 - many of these are similar to the options you see in the gene tab, but not all of them. If you can’t find the thing you’re looking for, often the solution is to switch tabs.  
 
 

Transcript sequences

Click on the Exons link in the left-hand panel. This page is useful as it will give you the length of the coding sequence.

You may want to change the display (for example, to show more flanking sequences). In order to do so, click on Configure this page and change the display options accordingly.  
 
 

Transcript information in external databases

Next, follow the General identifiers link at the left. Just like the External References page in the Gene tab, this page shows links out to other databases such as InterPro, PDB, UniProtKB, and others, this time linked to the transcript or protein product, rather than the gene.

Protein domain information

If you’re interested in protein domains, you could click on Protein summary to view domains from different sources, such as SMART and PROSITE. These are all plotted against the transcript sequence.

Alternatively, you can go to Domains & features to see a table of the same information in a tabular format.

Start in Ensembl Bacteria and select the Clostridium sporogenes (GCA_001444695) genome.

1. What GO: biological process terms are associated with the PolC gene?

2. Go to the transcript tab for the only transcript, OQP95999. How long is the transcript?

3. What domains can be found in the protein product of this transcript? How many different domain prediction methods agree with each of these domains?

Start in Ensembl Bacteria and search for the Escherichia coli str. K-12 substr. MG1655 (GCA_000005845) genome.

1. What GO: biological process terms are associated with the Era gene?

2. How many different InterPro domains are found in the protein product of this gene?

3. What is the associated UniProt ID of the transcript?

We’re going to look at the gene ATG8 in Magnaporthe oryzae in Ensembl Fungi. This gene is involved in autophagy, and targeted silencing of this gene inhibits infection (you can find further info in Wilson and Talbot, Nature Reviews Microbiology volume 7, pages 185–195 (2009)).

1. Find the genomic sequence of the M. oryzae ATG8 gene. How many exons does the gene have?

2. What is the GO accession ID for this gene?

3. How many transcripts does the gene have? Download the cDNA sequence in FASTA format to your computer.

4. Are there any entries of the gene in external databases? If so, which ones?

1. Find the Plasmodium falciparum PF3D7_1145400 gene on Ensembl Protists. On which strand is this gene located? What are the coordinates of the gene?

2. How long is its transcript (in bp)? How long is the protein it encodes? How many exons does it have?

3. What is the Uniprot ID that maps to the translation of this transcript?

4. What are the GO:Biological process(es) associated with PF3D7_1145400?

1. Find the Arabidopsis thaliana CCD7 gene on Ensembl Plants. On which chromosome and which strand of the genome is this gene located?

2. Where in the cell is the CCD7 protein located?

3. What is the source of the assigned gene name?

4. How many transcripts does it have? How long is its longest transcript (in bp)? How long is the protein it encodes? How many exons does it have? Are any of the exons completely or partially untranslated?

1. In Ensembl, find the human MYH9 (myosin, heavy chain 9, non-muscle) gene and open the Gene tab.
   • On which chromosome and which strand of the genome is this gene located?
   • How many transcripts (splice variants) are there and how many are protein coding?
   • What is the longest protein-coding transcript, and how long is the protein it encodes?
   • Which transcript would you take forward for further study?

2. Click on Phenotypes at the left side of the page. Are there any diseases associated with this gene, according to Mendelian Inheritance in Man (MIM)?

3. What are some functions of MYH9 according to the Gene Ontology (GO) consortium? Have a look at the GO: Biological process pages for this gene.

4. In the transcript table, click on the transcript ID for MYH9-201, and go to the Transcript tab.
   • How many exons does it have?
   • Are any of the exons completely or partially untranslated?
   • Is there an associated sequence in UniProtKB/Swiss-Prot? Have a look at the General identifiers for this transcript.
5. Are there microarray (oligo) probes that can be used to monitor ENST00000216181 expression?

Genetic variation in the dipeptidylpeptidase 6 Gene (DPP6) in humans has previously been strongly associated with amyotrophic lateral sclerosis (ALS), a lethal disorder caused by progressive degeneration of motor neurons in the brain.

1. Go to the Ensembl homepage, search for the Dpp6 gene in mouse and click on the transcript ID ENSMUST00000071500 to open the transcript tab. How many exons make up this transcript?

2. Click on Exons to display the exon sequences of the transcript. Which exon contains the translation start? What is the exon ID of the largest exon? What is the start and end phase of exon 2?

3. Go to the Protein summary. How many protein domains or features fall within the second exon? What is the Pfam protein domain at the C-terminus of the protein and how many exons does it fall into? Which amino acid positions does the domain above cover?

4. Go to Domains and features. Which domains are associated with Pfam? How many genes in the mouse genome have the IPR002469 domain? What chromosomes are these genes found on?

Demo: The gene tab

View all variants within a gene sequence

In any of the sequence views shown in the Gene and Transcript tabs, you can view variants on the sequence. You can do this by clicking on Configure this page from any of these views.

Let’s take a look at the Gene sequence view for ADH4 in Saccharomyces cerevisiae R64-1-1. Search for ADH4 and go to the Sequence view. If you can’t see variants marked on this view, click on Configure this page and select Show variants: Yes and show links.

Find out more about a variant by clicking on it.

You can add variants to all other sequence views in the same way.

You can go to the Variation tab by clicking on the variant ID. For now, we’ll explore more ways of finding variants.  
 
 

View all variants within a gene in tabular format

To view all the sequence variations in table form, click the Variant table link at the left of the gene tab

You can filter the table to only show the variants you’re interested in. For example, click on Consequences: All, then select the variant consequences you’re interested in.

You can also filter by other columns such as, SIFT or Source.  
 
 

Demo: The location tab

Visualise variants within a region

Let’s have a look at variants in the Location tab. Click on the Location tab in the top bar. Click Configure this page and open Variation from the left-hand menu.

There are various options for turning on variants. Turn on the Sequence variants (all sources) in Normal.

Click on a variant to find out more information. It may be easier to see the individual variants if you zoom in.  
 
 

Demo: The variant tab

Variant summary

Let’s have a look at a specific variant. The easiest way to find a specific variant is to search for it. Search for s07-16069 and click through to the Variant tab.

Variant consequences specific features

The icons show you what information is available for this variant. Click on Genes and regulation, or follow the link in the left-hand panel.

This variant is found in the YGL256W gene only.  
 
 

Genotype frequency

Let’s look at population genetics. Either click on Explore this variant in the left hand menu then click on the Genotype frequency icon, or click on Genotype frequency in the left-hand menu.

Individual sample genotypes

We can see which strains these genotypes were observed in by going to Sample Genotypes.

1. How many species in Ensembl Fungi have variation data?

2. Select Fusarium oxysporum (FO2) and search for the FOXG_13574T0 gene. One of its upstream variants is SNP tmp_10_6610. What are the possible alleles for this polymorphic position? Which one is on the reference genome?

3. What is the most frequent allele at this position?

4. Which samples have the genotypes C|T and T|T?

1. Go to Ensembl Plants and find the Solyc02g084570.3 gene in Solanum lycopersicum (tomato) and go to its Location tab. Can you see the variation track?

2. Zoom in around the last exon of this gene. What are the different types of variants seen in that region? Are any splice region variants mapped in the region? If so, what is/are the coordinate(s)?

The SNP rs1738074 in the 5’ UTR of the human TAGAP gene has been identified as a genetic risk factor for a few diseases. Use Ensembl to answer the following questions:

1. In which transcripts is this SNP found?

2. What is the least frequent genotype for this SNP in the Yoruba (YRI) population from the 1000 Genomes phase 3?

3. What is the ancestral allele? Is it conserved in the 91 eutherian mammals EPO-Extended?

4. With which diseases is this SNP associated? Are there any known risk (or associated) alleles?

In the paper “Altered metabolic signature in pre-diabetic NOD mice” (PloS One. 2012; 7(4): e35445), Madsen et al. have described several regulatory and coding SNPs, some of them in genes involved in ATP and adenosine metabolism, leading to potentially faulty metabolism of ATP and adenosine. The authors describe that one of the identified SNPs in the murine Entpd2 gene (rs28232063) would lead to increased amounts of available ATP, an immune activator, causing increased cell activation and possibly autoreactive T-cell activation. Use Ensembl to answer the following questions:

1. Where is the SNP located (chromosome and coordinates)?

2. What is the HGVS recommendation nomenclature for this SNP?

3. Why does Ensembl put the G allele first (G/A)?

4. Are there differences between the genotypes reported in C57BL/6NJ and NOD/ShiLtJ, according to the Mouse Genomes Project?

Demo: VEP

Input

We have identified 5 variants on Saccharomyces cerevisiae R64-1-1 chromosome VII:
T -> C at 3598
G -> C at 3929
T -> G at 5566
T -> A at 5727
A -> T at 7628

We will use Ensembl VEP to determine:

• Have my variants already been annotated in Ensembl?
• What genes are affected by my variants?
• Are any of my variants missense variants?

Click on Tools in the navigation bar at the top of any Ensembl Fungi page, then click Variant Effect Predictor to open the input form:

Click on Add/remove species and search for Saccharomyces cerevisiae R64-1-1 to select it.

First, we need to convert our variants into one of the formats supported by VEP. You can find a list of input formats and examples in the VEP documentation page. Let’s put our data in VCF:
chromosome coordinate id reference alternative

Copy and paste the following data into the Input data box:

VII 3598 var1 T C
VII 3929 var2 G C
VII 5566 var3 T G
VII 5727 var4 T A
VII 7628 var5 A T

VEP will automatically detect that the data is in VCF.  
 
 

Additional configurations

There are further options that you can choose for your output. These are categorised as Identifiers, Variants and frequency data, Additional annotations, Predictions, Filtering options and Advanced options. You can open all tabs to explore the different options.

Hover over the options to see definitions. When you’ve selected everything you need, scroll right to the bottom and click Run.

The display will show you the status of your job. It will say Queued, then automatically switch to Done when the job is done, you do not need to refresh the page. You can edit or discard your job at this time. If you have submitted multiple jobs, they will all appear here.

Click View results once your job is done.  
 
 

Results

In your results you will see a graphical summary of your data, as well as a table of your results.

The results table is very detailed, so we’re going to go through the it by section. The first column is Uploaded variant. If your input data contains IDs, like ours does (i.e. var1, var2, etc.), the ID is listed here. If your input data is only loci, this column will contain the locus and alleles of the variant. You’ll notice that the variants are not neccessarily in the order they were in in your input. You’ll also see that there are multiple lines in the table for each variant, with each line representing one transcript or other feature the variant affects.

You can hover over any column name with your mouse to get a definition of what is shown.

The next few columns show information about the feature the variant affects, including the consequence. Where the feature is a transcript, you will see the gene symbol and stable ID and the transcript stable ID and biotype. The IDs are links to take you to the Gene or Transcript tabs.

This is followed by details on the effects on transcripts, including the position of the variant in terms of the exon number, cDNA, CDS and protein, the amino acid and codon change and pathogenicity scores. Where the variant is known, the ID of the existing variant is listed under the column Existing variant, with a link out to the Variant tab. The pathogenicity scores are shown as numbers with coloured highlights to indicate the prediction, and you can mouse-over the scores to get the prediction in words.

Above the table is the Filter option, which allows you to filter by any column in the table. You can select a column from the drop-down, then a logic option from the next drop-down, then type in your filter to the following box. We’ll try a filter of Consequence, followed by is then missense_variant, which will give us only variants that change the amino acid sequence of the protein. You’ll notice that as you type missense_variant, the VEP will make suggestions for an autocomplete.

You can export your VEP results in various formats, including VCF. When you export as VCF, you’ll get all the VEP annotation listed under CSQ in the INFO column of the VCF file. After filtering your data, you’ll see that you have the option to export only the filtered data.

In Ensembl Bacteria the genome for Bacteroides fragilis 638R and launch the VEP tool. Use VEP to predict the effects of a 7 bp deletion of TCTACAA on the supercontig FQ312004 at the position 258140-258146. Use the results to answer the following questions:

1. How many genes does the indel overlap? What are their gene symbols?

2. What is the most common consequence of the variant?

3. What is the most severe consequence? What gene does it affect and what does it do?

We have identified four variants in Verticillium dahliae JR2 chromosome 5:

• C->G at 698711
• G->T at 698935
• G->A at 700313
• C->A at 701484

Use VEP in Ensembl Fungi to answer the following questions:

1. Have these variants already been annotated in Ensembl?

2. What genes are affected by the variants? What are their gene IDs?

3. Are any of the variants predicted to be missense variants?

You’ll find a VCF file here. This is a small subset of the outcome of Oryza sativa Japonica whole-genome sequencing and variant-calling experiment. Analyse the variants in this file with the VEP tool in Ensembl Plants and determine the following:

1. How many genes and transcripts are affected by variants in this file?

2. Do these variants result in a change in the proteins encoded by any of the Ensembl genes? Which genes are affected? What is the amino acid change?

Below, you will find a list of variants which have been reported in the FLG gene (ENSG00000143631), which codes for filaggrin. Mutations in the gene have been associated with atopic dermatitis.

• C/A at 1: 152,302,797
• G/A at 1: 152,307,085
• C/T at 1: 152,310,208
• G/A at 1: 152,308,234
• C/T at 1: 152,313,454
• C/T at 1: 152,309,920
• A/G at 1: 152,309,268

Use the VEP tool in Ensembl and find out the following information:

1. What consequences have been predicted for the variants?

2. Are SIFT and PolyPhen predictions available for the variants? What are the scores?

3. How many of these variants have been previously reported?

We have details of a genomic deletion in a breast cancer sample in VCF format:

13 32307062 sv1 . <DEL> . . SVTYPE=DEL;END=32908738

Use VEP in Ensembl to find out the following information:

1.  How many genes have been affected?

2.  Does the structural variant (SV) cause deletion of any complete transcripts?

3.  Map your variant in the Ensembl browser on the Region in detail view.

Demo: gene trees and homology predictions

Fungal Compara

Gene trees

Let’s look at the homologues of Saccharomyces cerevisiae R64-1-1 TAZ1 (gene stable ID: YPR140W). This gene is involved in stress response and conserved across different taxonomic domains. Search for the gene and go to the Gene tab.

Click on Fungal Compara: Gene tree. This will display the current gene in the context of a phylogenetic tree used to determine orthologues and paralogues.

Funnels indicate collapsed nodes. Click on a node (coloured triangle) to open a menu. We can then see what type of node this is, some statistics and options to expand or export the sub-tree.

There are some quick filtering options below the image, where you can add paralogues, and quickly expand or collapse nodes.

You can download the tree in a variety of formats. From the pop-up above you can click to export the sub-tree (everything to the right of the node). Alternatively, click on the Export icon in the bar at the top of the image to get a pop-up where you can choose your format. You can preview this file before you download.

Homologues

We can look at homologues in the Orthologues and Paralogues pages, which can be accessed from the left-hand menu. If there are no orthologues or paralogues, then the name will be greyed out. Click on Fungal Compara: Orthologues to see the orthologues available. In the first table, you will find a summary of orthologues by taxonomic group:

In the second table, you will find orthologue details per species:

Scroll to the bottom of the page to see a list of the species that do not have any orthologues with TAZ1 in S. cerevisiae… there are a lot!

Pan-taxonomic Compara

S. cerevisiae is part of Pan-compara, which compares a subset of fungal species with species from other taxa, such as plants, bacteria and vertebrates. Click on Pan-taxonomic Compara: Orthologues. Let’s see if there are any orthologues of TAZ1 in plants. Click the Show details box for Plants.

Demo: Whole-genome alignments

Alignments in the Region in Detail view

Let’s look at some of the comparative genomics views in the Location tab. Go to the region 2:438432-458358 in Fusarium solani. This region includes a number of genes and we want to find out if any regions align with Fusarium verticillioides. To do this, we need to look at a pairwise alignment between the two Fusarium species. We can look at individual species comparative genomics tracks in this view by clicking on Configure this page. In the Comparative genomics section, turn on the F. verticillioides track in the normal format.

We can now see some pink alignments shown on the display. Alignments to the same chromosome are presented in a single row, and gaps in the alignment are shown by empty blocks. If there are alignments to multiple chromosomes in the aligned species, they are represented on different rows. From the track, we can see that the regions encoding genes NechaG90679, NechaG57836, NechaG26181, NechaG32512 and NechaG75968 align perfectly between the two genomes.

Sequence alignments

We can also look at the sequence alignment between the two species as text. Click on Comparative Genomics: Alignments (text) in the left-hand menu.

Click on Select an alignment to select a species you want to align. Let’s select F. solani and click Go. There are two blocks aligned of different lengths, some of which correspond to the region we just saw in the Region in detail view.

Click on Block 1.

You will see a list of aligned regions, followed by the sequence alignment. Click on Display full alignment. Exons are shown in red. Click on Configure this page on the left. In the pop-up menu, you can turn on the options to view Show conservation regions and Mark alignment start/end. This will add highlights where the sequence matches.  
 
 

Region comparison

To compare the two genomic regions visually, go to Region Comparison in the left-hand panel. To add species to this view, click on the Select species or regions button. Select F. verticillioides again then close the menu. This page, similar to the Region in detail view, shows the chromosome positions first. We can see the location of this alignment on chromosome 2 in F. solani. You can scroll down to the most detailed image to view aligned regions, which are highlighted and linked in green.

You can add data to both of these views with the same options you had in the Region in detail page. Click on Configure this page to open the menu.  
 
 

Synteny

We can view large-scale syntenic regions from our chromosome of interest. Click on Synteny in the left-hand panel. Black linking lines indicate sequences are oriented in the same directed, red linking lines indicate the sequences are inverted.

Bacillus subtilis subsp. subtilis str. 168 (GCA_000009045) is a model organism and often used in academic research and in the biotechnology industry as it can produce large amounts of important enzymes, like protease and amylase. It is part of Pan-taxonomic Compara in Ensembl Bacteria. We will use the sipT gene, a type I signal peptidase, as a reference to find the following information:

1. Find the Ensembl gene tree ID. How many speciation and duplication nodes does it have?

2. How many orthologues does B. subtilis str. 168 have? What type of orthologues are they?

3. Does it have an orthologue in Escherichia coli str. K-12 substr. MG1655? If so, what is the gene ID and coordinate in E. coli?

4. Export the protein alignment of the B. subtilis and E. coli orthologues. What are the different formats you can export the alignment as?

Go to the Ensembl Fungi site to find out the following:

1. How many orthologues and how many paralogues are predicted for the Schizosaccharomyces pombe Mcm6 gene?

2. How many Schizosaccharomyces orthologues are there?

3. Does it have a human orthologue? If so what type is it and what is the orthologue’s Ensembl ID?

The fumarase gene FUM1 in Arabidopsis thaliana encodes a protein with mitochondrial targeting information. Read more in this UniProt entry. Go to Ensembl Plants to answer the following questions:

1. How many orthologues have been identified for this gene?

2. Which orthologue has the highest sequence similarity? Look at the Query%ID and Target%ID.

Search Ensembl Plants for the gene Lsi1 in Oryza sativa Japonica (rice). This gene is known to code for an aquaporin transporter that facilitates the uptake of silicon and arsenic through the roots. Silicon concentration is highest in grass species, and is associated with defence.

1. From the gene tab, go to the Orthologues page under Plant Compara. Which plant group has the highest number of 1-to-1 orthologues? Is it the same group that has the highest number of 1-to-many orthologues?

2. Reduce the orthologues table to look only at Triticum aestivum (wheat) orthologues. Why are there three results for a 1-to-1 orthologue?

3. Click on the Compare regions link for chromosome 6B region in wheat to go to the Location tab. Scroll to the bottom image. How do the gene models compare between the species? Do they have the same number of exons?

4. Click back to the Gene tab and click on the Gene gain/loss tree page. Which species has the highest number of members of this gene family? Is it a grass? Can you change the view to see a radial tree?

Go to Ensembl to answer the following questions:

1. How many orthologues are predicted for the human BRAF in primates? How much sequence identity does the Carlito syrichta (tarsier) protein have to the human one? Can you tell which end of the BRAF protein is more conserved between these two species by looking at the orthologue alignment?

2. Go to the Gene tree for this gene. View the Wasabi alignment of all the proteins in primates. Can you see a large gap in the alignment around position 450? Which species match the human sequence?

Start at Ensembl homepage.

1. Find the rhodopsin (RHO) gene in human. Go to the Location tab and click Synteny at the left. Are there any syntenic regions in duck? If so, which chromosomes are shown in this view?

2. Stay in the Synteny view. Is there a homologue in duck for human RHO? Are there more genes in this syntenic block with homologues? Which duck chromosome is this human genomic region syntenic to?

You find a deletion in the mouse genome that severely affects development, and you want to investigate it. In Ensembl, go to this region (base pair coordinates 136197000-136283000 on mouse chromosome 4).

1. How many protein coding transcripts does the gene in this region have?

2. You want to see if any of these transcripts might be expressed in brain tissue. Turn on the RNASeq gene model for brain.

3. Find this same view (Region in Detail) for the human orthologue of the mouse gene.

4. To understand crucial variants, you want to investigate if there are any missense variants in the most diverse population available. Turn on the 1000 Genomes AFR (African population) track.

Demo: BioMart

Follow these instructions to guide you through BioMart to answer the following queries:

1. How many genes are found in Fusarium solani that do not have an orthologue in Fusarium oxysporum?
2. How many of these are associated with a pathogenic phenotype of “reduced virulence”?
3. Export the gene name, locations and GO terms associated with these genes
4. Export their cDNA sequences  
    
    

Step 1: Choose database and dataset

Click on BioMart located on the navigation bar at the top of any fungi.ensembl.org page.

Step 2: Choose appropriate filters

We want to find all genes in F. solani that do not have an orthologue with F. oxysporum. We need to filter the dataset to look only at these genes.

Using the count function we can see that there are 6,137 F. solani genes (out of a total of 16,163) that do not have an orthologue in F. oxysporum. We also want to find out how many of these are associated with the PHI-base Pathogen phenotype ‘reduced virulence’.

We can now see from the count information that we have 3 genes associated with this phenotype that do not have any orthologues with F. oxysporum.  
 
 

Step 3.1: Select attributes (features)

Attributes are defined by what we would like to learn about the data. We want to find out more information about the genes:

1. Gene name
2. Locations
3. Associated GO terms
4. cDNA sequences

You can only select one attribute category at a time. We can answer points 1-3 in a single query, but we will need to do a second query to answer point 4. Make sure that Features category is selected at the top of the page. Expand the GENE tab and select the following features:

• Gene Name
• Chromosome/scaffold name
• Gene start (bp)
• Gene end (BP)

Next, expand the EXTERNAL tab. This section contains lots of identifiers from databases outside of Ensembl. Select the following features:

• GO term accession
• GO term name

Step 4: Get the results

You can download the data if you desire. The table presented shows a sub-sample of 10 results to enable you to check you have the correct attributes.

We can see that all of these genes are located in the same region. Perhaps this has something to do with why they are not found in F. oxysporum…? You can click on the location links and explore the synteny between the two species.  
 
 

Step 3.2: Select attributes (sequences)

To complete the fourth part of the demo, we need to export the cDNA sequences of our three genes. To do this, go back to the Attributes section in the left-hand panel and select Sequences from the attributes categories at the top of the page.

Also expand the HEADER INFORMATION tab and select Gene name. Click Results in the left-hand panel to see your sequences.

For more details on BioMart, have a look at this publication: Kinsella RJ, Kähäri A, Haider S, et al. Ensembl BioMarts: a hub for data retrieval across taxonomic space. Database : the Journal of Biological Databases and Curation. 2011 ;2011:bar030. DOI: 10.1093/database/bar030. PMID: 21785142; PMCID: PMC3170168.

In Ensembl Fungi, retrieve the variation ID, chromosome name, position in bp and alleles for all sequence variants in the mtDNA. Export the results as an XLS table and have the results sent to you by email.

Go to Ensembl Protists. For a list of , export the Find Leishmania major orthologues for these Trypanosoma brucei genes: Tb927.3.3470, Tb927.3.3520, Tb11.01.7770, Tb927.8.5110, Tb927.1.1420

One class of disease resistance (R) genes in plants are the TIR-NBS-LRR genes, that code for proteins that contain an N-terminal Toll/Interleukin receptor homology region (TIR), a nucleotide binding site (NBS) and a C-terminal leucine rich repeat (LRR). TIR-NBS-LRR genes are common in dicots but seem to be rare in monocots (Tarr and Alexander. TIR-NBS-LRR genes are rare in monocots: evidence from diverse monocot orders. BMC Res Notes 2009 Sep 8;2:197).

The ID for the TIR domain in the Pfam (protein family) database is PF01582.

Use BioMart in Ensembl Plants to generate a list of all Solanum tuberosum (potato; a dicot) genes that are annotated to contain a TIR domain. Include the Ensembl stable ID and gene description. Do the same for Zea mays (maize; a monocot).

Do your results confirm the findings of Tarr and Alexander?

BioMart is a very handy tool when you want to map between different databases. The following is a list of IDs from the UniProtKB/Swiss-Prot database of Arabidopsis thaliana proteins that are supposedly involved in flavonoid metabolism: P42813, Q9LS08, Q9ZST4, Q9SYM2, P51102, Q9LPV9, Q9FE25, Q96323, Q9FKW3, P13114, P41088, Q9S818, Q96330, O22203, Q39224, O22264, Q9SD85, Q9LYT3, Q9FJA2, Q43128, P43254, O04153, Q43125, Q9S9P6, Q94C57, Q9LNE6, Q9FK25, Q9SYM5, Q9ZQ95

Using BioMart in Ensembl Plants a list that shows to which Ensembl Gene IDs these UniProtKB/Swiss-Prot IDs map. Also include the gene name and description.

BioMart is a very handy tool when you want to convert IDs from different databases. The following is a list of 29 IDs of human proteins from the NCBI RefSeq database:
NP_001218, NP_203125, NP_203124, NP_203126, NP_001007233, NP_150636, NP_150635, NP_001214, NP_150637, NP_150634, NP_150649, NP_001216, NP_116787, NP_001217, NP_127463, NP_001220, NP_004338, NP_004337, NP_116786, NP_036246, NP_116756, NP_116759, NP_001221, NP_203519, NP_001073594, NP_001219, NP_001073593, NP_203520, NP_203522

Use BioMart in Ensembl to generate a list that shows to which Ensembl gene IDs and to which gene names these RefSeq IDs correspond. Do these 29 transcripts correspond to 29 genes?

Go to Ensembl’s BioMart. For a list of Ciona savignyi Ensembl genes, export the human orthologues:
ENSCSAVG00000000002, ENSCSAVG00000000003, ENSCSAVG00000000006, ENSCSAVG00000000007, ENSCSAVG00000000009, ENSCSAVG00000000011

Do all of these genes have a homologue in human?