Demo: Introduction to Ensembl Plants

Homepage

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

At the top left you can see the current release number and what has come out in this release.

Available species

Click on View full list of all species.

Click on the scientific name of your species of interest to go to the species homepage. We’ll click on Coffea canephora.

Species information

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

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

1. What is the name of the coffee variety represented in Ensembl?

2. Who produced this genome assembly and annotation?

3. What is the length of the Coffea canephora genome assembly? How many coding genes are annotated across the genome?

Botrytis cinerea is the causal agent of the grey mold disease and warty berry in coffee.

1. Who produced this genome assembly and annotation?

2. What is the length of the Botrytis cinerea genome assembly? How many coding genes are annotated across the genome?

Demo: Region in Detail view

Start at the Ensembl Plants front page. You can search for a region by typing it into a search box, but you have to specify the species.

To bypass the text search, you need to input your region coordinates in the correct format, which is chromosome, colon, start coordinate, dash, end coordinate, with no spaces for example: 6:9093257-9115373. Choose _Coffea canephora (AUK_PRJEB4211_v1) _ from the species drop-down, then type (or copy and paste) these coordinates into the 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 in detail.

1. The first image shows the chromosome:

The region we’re looking at is highlighted on the chromosome.

1. The second image shows a 1Mb region around our selected region. This is always 1Mb in human, but the fixed size of this view varies between species. This view allows you to scroll back and forth along the chromosome.

You can also drag out and jump to or mark a 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 two blue buttons appear. Clicking on Update this image would jump the lower image to the region central to the scrollable image. We want to go back to where we started, so we’ll click on Reset scrollable image.

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

Here you can see various tracks, which is what we call a data type that you can plot against the genome. Some tracks, such as the transcripts, can be on the forward or reverse strand. Forward stranded features are shown above the blue contig track that runs across the middle of the image, with reverse stranded features below the contig. Other tracks, such as variants, regulatory features or conserved regions, refer to both strands of the genome, and these are shown by default at the very top or very bottom of the view.

You can use click and drag to either navigate around the region or highlight regions of interest, 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.

With the tool set to Select, drag out a box around an exon and choose Mark region.

The highlight will remain in place if you zoom in and out or move around the region. This allows you to keep track of regions or features 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.

There are thousands of possible tracks that you can add. When you launch the view, you will see all the tracks that are currently turned on with their names on the left and an info icon on the right, which you can click on to expand the description of the track. Turn them on or off, or change the track style by clicking on the box next to the name. More details about the different track styles are in this FAQ.

You can find more tracks to add by either exploring the categories on the left, or using the Find a track option at the top left. Type in a word or phrase to find tracks with it in the track name or description.

Let’s add a track to this image. Add:

• All Repeats

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

If the track is not giving you can information you need, you can easily change the way the tracks appear by hovering over the track name then the cog wheel to open a menu. To make it easier to compare information between tracks, such as spotting overlaps, you 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 link. 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 Ensembl Plants.

1. Go to the region from 23,704,000 to 23,766,000 bp on coffee chromosome 1.

2. Zoom in on the GSCOC_T00030044001 gene with transcript ID CDP09644.

3. Configure this page to turn on the Repeats (Repbase) track in this view. What tool was used to annotate the repeats according to the track information? How many repeats can you see within the GSCOC_T00030044001 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.

You can find out lots of information about Ensembl genes and transcripts using the browser. If you’re already looking at a region 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 or identifiers, and also phenotypes or functions that might be associated with the genes.

We’re going to look at the Coffea canephora GSCOC_T00022371001 gene. From plants.ensembl.org, type GSCOC_T00022371001 into the search bar and click the Go button.

The gene tab

Click on GSCOC_T00022371001 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 view with the transcripts. We can see exons shown as blocks with introns as lines linking them together. Coding exons are filled, whereas non-coding exons are empty. We can also see the overlapping and neighbouring genes and other genomic features.

There are different tabs for different types of features, such as genes, transcripts or variants. These appear side-by-side across the blue bar, 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.

Let’s walk through this menu for the gene tab. How can we view the genomic sequence? Click Sequence at the left of the page.

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, both exons of our gene of interest and any neighbouring or overlapping gene. By default, 600 bases are shown up and downstream of the gene. We can make changes to how this sequence appears with the blue 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.

Once you have selected changes (in this example, Line numbering) click at the top right.

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 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.

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

To find the biochemical pathways that this gene is involved in, from Reactome, click on Pathway in the left-hand menu

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 Expression Atlas or UniProtKB. Go up the left-hand menu to External references:

Demo: The transcript tab

We’re now going to explore the different transcripts of GSCOC_T00022371001. Click on Show transcript table at the top.

Here we can see that there is only one transcript of GSCOC_T00022371001 with its identifier, length and biotype. Click on the ID of the Ensembl Canonical transcript, CDO98315.

You are now in the Transcript tab for CDO98315. We can still see the gene tab so we can easily jump back. The left hand navigation column provides several options for the transcript CDO98315 - 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.

Click on the Exons link. This page is useful for designing RT-PCR primers because you can see the sequences of the different exons and their lengths.

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

Now click on the cDNA link to see the spliced transcript sequence with the amino acid sequence. This page is useful for mapping between the RNA and protein sequences, particularly genetic variants.

UnTranslated Regions (UTRs) are highlighted in dark yellow, codons are highlighted in light yellow, and exon sequence is shown in black or blue letters to show exon divides. Sequence variants are represented by highlighted nucleotides and clickable IUPAC codes are above the sequence.

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 RefSeq, UniProtKB, PDBe and others, this time linked to the transcript or protein product, rather than the gene.

If you’re interested in protein domains, you could click on Protein summary to view domains from Pfam, PROSITE, Superfamily, InterPro, and more. These are all plotted against the transcript sequence, with the exons shown in alternating shades of purple at the top of the page. Alternatively, you can go to Domains & features to see a table of the same information.

You can also view the AlphaFold predicted 3D structure of the protein. Click on AlphaFold predicted model in the left-hand menu.

The FUS3 gene is a known master regulator of somatic embryogenesis, an important factor in stable genetic transformation and successful plant regeneration of coffee trees expressing the Bacillus thuringiensis (Bt) toxin Cry10Aa to induce Coffee Berry Borer (CBB) resistance.

1. Find the Coffea canephora FUS3 gene on Ensembl Plants. On which chromosome and which strand of the genome is this gene located?

2. Where in the cell is the FUS3 protein located?

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

4. How long is its 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?

Rosellinia necatrix is is a fungal plant pathogen infecting several hosts including coffee, apples, apricots, avocados, cassava, strawberries, pears, hop, citruses and Narcissus, causing white root rot. A study by A. Zumaquero et al in 2019 (doi: 10.1186/s12864-019-6387-5) revealed SAMD00023353_4000440 as a gene potentially involved in pathogenesis.

Start in Ensembl Fungi and select the Rosellinia necatrix str. W97 (GCA_001445595) genome.

1. What GO: molecular function terms are associated with the SAMD00023353_4000440 gene?

2. Go to the transcript tab for the only transcript, GAP89412. 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?

Exploring variants in Arabidopsis thaliana

Visualising variants in the Sequence view

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 PAD4 in Arabidopsis thaliana. Search for PAD4 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.

Viewing variants within a gene in the tabular form

To view all the sequence variants 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. For display purposes, the table above has already been filtered to only show missense variants.

You can also filter by the different pathogenicity scores and MAF, or click on Filter other columns for filtering by other columns such as Evidence or Class.

The table contains lots of information about the variants. You can click on the IDs here to go to the Variation tab too.

Visualising variants in the Region in Detail view

Let’s have a look at variants in the Location tab. Click on the Location tab in the top bar.

Configure this page and open Variation from the left-hand menu.

There are various options for turning on variants. You can turn on variants by source or presence of a phenotype. You can also turn on genotyping chips.

Turn on Sequence Variants (all sources) in Normal format.

Exploring a specific variant

Let’s have a look at a specific variant. If we zoomed in we could see the variant tmp_319431818_G_C in this region, however it’s easier to find if we put _tmp_3_19431818_G_C into the search box. Click through to open the Variation tab for Arabidopsis thaliana.

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

This page illustrates the genes the variant falls within and the consequences on those genes, including pathogenicity predictors.

Let’s look at population genetics. Click on Population genetics in the left-hand menu.

The population allele frequencies are shown by study. Where genotype frequencies are available, these are shown in the tables.

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

The Arabidopsis thaliana ATCDSP32 protein is a chloroplastic drought-induced stress protein proposed to participate in a process called cell redox homeostasis. Go to Ensembl Plants and answer the following questions:

1. How many variants have been identified in the gene that can cause a change in the protein sequence (i.e. missense variant)?

2. What is the ID of the variant that changes the amino acid residue 60 from Alanine to Threonine (hint: refer to an amino acid codon table)? What is the location of this SNP in the A. thaliana genome? What are its possible alleles?

3. Download the flanking sequence of this SNP in RTF (Rich Text Format). Can you change how much flanking sequence is displayed on the browser?

4. Does this SNP cause a change at the amino acid level for other genes or transcripts?

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)?

Demonstration of the VEP web interface

Input

We have identified three variants on coffee chromosome 4:
T -> C at 25759812
G -> A at 25685623
T -> G at 25863697

We will use the Ensembl VEP to determine which genes are affected by my variants?

Click on Tools in the top green bar from any Ensembl Plants page, then Variant Effect Predictor to open the input form:

Click on Add/remove species and search for Triticum aestivum to choose it.

The data is in VCF:
chromosome coordinate id reference alternative

Put the following into the Paste data box:

4 25759812 var1 T C  
4 25685623 var2 G A
4 25863697 var3 T G

The 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. Let’s open all the menus and take a look.

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 enormous and 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, 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 mouse over any column name to get a definition of what is shown.

The next few columns give the 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 homepage.

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, as well as the amino acid and codon change.

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. After filtering your data, you’ll see that you have the option to export only the filtered data. You can also drop all the genes you’ve found into the Gene BioMart, or all the known variants into the Variation BioMart to export further information about them.

You have done whole-genome sequencing and variant-calling experiments for Coffea canephora. You have a VCF file with a small subset of variants from this experiment. Analyse the variants in this file with the VEP tool in Ensembl Plants and determine the following:

1 35246062 var1 C A  
1 35246078 var2 G C
1 35246154 var3 T A

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

2. Filter the table to find variants with high impact. How many variants have high impact and what consequence predictions do they refer to? Why do you think missense variants are not classified as high impact?

3. Can you export all the results to a VCF file? Compare it to the input VCF file to see what information the VEP adds.

Demo: gene trees and homology predictions

Plants Compara

Gene trees

Let’s look at the homologues of Coffea canephora (coffee) GSCOC_T00022371001. Open Ensembl Plants, search for the gene and go to the Gene tab.

Click on Plant compara: Gene tree, which will display the current gene in the context of a phylogenetic tree used to determine orthologues and paralogues.

Funnels indicate collapsed nodes. We can expand them by clicking on the node and selecting Expand this sub-tree from the pop-up menu.

We can also see the protein alignment of the sub-tree by clicking on Wasabi viewer, which will open a pop-up:

You can download the tree in a variety of formats. Click on the download icon in the bar at the top of the image to get a pop-up where you can choose your format.

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 Plant compara: Orthologues to see the orthologues available in plants.

Choose to see only Eudicotyledons orthologues by selecting the box. The table below will now only show details of Eudicotyledons orthologues. Let’s look at Arabidopsis thaliana.

Here we can see there is a many-to-many relationship between the coffee and A. thaliana orthologues. Links from the orthologue allow you to go to alignments of the orthologous proteins and cDNAs. Click on View Sequence Alignments then View cDNA Alignment for the first A. thaliana orthologue.

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 4:4:4640000-4680000 in Coffea canephora (coffee). We can look at individual species comparative genomics tracks in this view by clicking on Configure this page.

Select Comparative genomics from the left-hand menu to choose alignments between closely related species. Turn on the alignments for Arabidopsis thaliana, Oryza sativa japonica Group (rice) and Solanum lycopersicum (tomato).

Sequence alignments

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

Click on Select an alignment to open the alignment menu. Select A. thaliana from the alignments list then click Go.

In this case there are 12 blocks aligned of different lengths. Click on Block 1.

You will see a list of the regions aligned, followed by the sequence alignment. Click on Display full alignment. Exons are shown in red (you may need to scroll down the page to see the first exon).

Region comparison

To compare with both contigs visually, go to Comparative Genomics: Region Comparison.

To add species to this view, click on the green Select species or regions button. Choose A. thaliana again then close the menu.

Synteny

We can view large-scale syntenic regions from our chromosome of interest. Click on Comparative Genomics: Synteny in the left-hand menu and select S. lycopersicum* from the **Change species drop-down in the right-hand side.

Black linking lines indicate sequences are oriented in the same directed, red linking lines indicate the sequences are inverted.

Resistance to the leaf rust delivered by SH3 factor(s) is well-grounded as specially durable. in 2023, Paula Cristina da Silva Angelo et al (https://doi.org/10.1016/j.pmpp.2023.102111) reported that the Arabidopsis thaliana gene AT1G50180 is an important gene in the SH3 locus conferring diseae resistance.

Search Ensembl Plants for the gene AT1G50180 in Arabidopsis thaliana.

1. From the gene tab, go to the Arabidopsis thaliana AT1G50180 gene Orthologues page under Plant Compara.

2. Reduce the orthologues table to look only at Coffea canephora (coffee) orthologues. How many results can you see?

3. Download the cDNA alignment in ClustalW format for the alignment between the Arabidopsis thaliana AT1G50180 gene and the Coffea canephora GSCOC_T00030728001 gene.

Demo: BioMart

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

1. What genes are found on chromosome 6, between 8950000 and 8999000 in coffee?
2. What is the transcript length (UTR + CDS)?
3. Are there associated functions from the GO (gene ontology) project that might help describe their function?
4. What are their cDNA sequences?

Step 1: Choose the database and dataset

Click on BioMart in the top header of any Ensembl Plants page to open BioMart

Step 2: Choose appropriate filters

Step 3.1: Select attributes (features)

Step 4.1: Get the results

Why are there multiple rows for one gene ID? For example, look at the first few rows.

Step 3.2: Select attributes (sequences)

Step 4.2: Get the results

Note: you can use the Go button to export a file.

What did you learn about the coffee genes in this exercise?

Could you learn these things from the Ensembl browser? Would it take longer?

Find Coffea canephora (coffee) proteins with Signalp cleavage sites located on chromosome 7.

Go to Ensembl Plants’s BioMart. For a list of Arabidopsis thaliana genes, export the coffee orthologues:
MLP28, MEE18, EP1, QRT3, MOT2, GC4, WYR

Do all of these genes have a homologue in coffee?