We have identified five variants on human chromosome nine, C-> A at 128203516, an A deletion at 128328461, C->A at 128322349, C->G at 128323079 and G->A at 128322917.

We will use the Ensembl VEP to determine:

• Have my variants already been annotated in Ensembl?
• What genes are affected by my variants?
• Do any of my variants affect gene regulation?

Go to the front page of Ensembl and click on the Variant Effect Predictor.

This page contains information about the VEP, including links to download the script version of the tool. Click on Launch VEP to open the input form:

The data is in VCF format:
chromosome coordinate id reference alternative

Put the following into the Paste data box:
9 128328460 var1 TA T
9 128322349 var2 C A
9 128323079 var3 C G
9 128322917 var4 G A
9 128203516 var5 C A

The VEP will automatically detect that the data is in VCF.

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.

We’re going to select some options:

• HGVS, annotation of variants in terms of the transcripts and proteins they affect, commonly-used by the clinical community
• Phenotypes
• Protein domains

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.

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. Where the feature is a regulatory feature, you will get the stable ID and type. For a transcription factor binding motif (labelled as a MotifFeature) you will see just the ID. Most of the IDs are links to take you to the gene, transcript or regulatory feature 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, the amino acid and codon change, transcript flags, such as MANE, on the transcript, which can be used to choose a single transcript for variant reporting, and predicted pathogenicity scores. The predicted 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. Two options that we selected in the input form are the HGVS notation, which is shown to the left of the image below and can be used for reporting, and the Domains to the right. The Domains list the proteins domains found, and where there is available, provide a link to the 3D protein model which will launch a LiteMol viewer, highlighting the variant position.

Where the variant is known, the ID of the existing variant is listed, with a link out to the variant homepage. In this example, only rsIDs from dbSNP are shown, but sometimes you will see IDs from other sources such as COSMIC. The VEP also looks up the variant from frequency files, and pulls back the allele frequency (AF in the table), which will give you the 1000 Genomes Global Allele Frequency. In our query, we have not selected allele frequencies from the continental 1000 Genomes populations or from gnomAD, but these could also be shown here. We can also see ClinVar clinical significance and the phenotypes associated with known variants or with the genes affected by the variants, with the variant ID listed for variant associations and the gene ID listed for gene associations, along with the source of the association.

For variants that affect transcription factor binding motifs, there are columns that show the effect on motifs (you may need to click on Show/hide columns at the top left of the table to display these). Here you can see the position of the variant in the motif, if the change increases or decreases the binding affinity of the motif and the transcription factors that bind the motif.

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.

Resequencing of the genomic region of the human CFTR (cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7) gene (ENSG00000001626) has revealed the following variants. The alleles defined in the forward strand:

• G/A at 7: 117,530,985
• T/C at 7: 117,531,038
• T/C at 7: 117,531,068

Use the VEP tool in Ensembl and choose the options to see SIFT and PolyPhen predictions. Do these variants result in a change in the proteins encoded by any of the Ensembl genes? Which gene? Have the variants already been found?

7	117530985	117530985	G/A
7	117531038	117531038	T/C  
7	117531068	117531068	T/C

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.

Introduction
The Ensembl Variant Effect Predictor (VEP) is a powerful tool for annotating genomic variants. Ensembl VEP is accessible via web, REST API and command-line options.

In this practical session, we will practice using Ensembl VEP via the web interface to annotate variants in HGVS format. The variants we will use were from an assay of exon 10 in the Homo sapiens BAP1 gene from the MaveMD collection on MaveDB. You can access the data on MaveDB here via the Scores file.

Ensembl VEP tutorials
A general Ensembl VEP tutorial for the web interface is available on the EMBL-EBI Training website to try out or compare to if you need some guidance. You can use the Ensembl VEP web documentation pages to help find and understand the configurations you will need.

Launch the Ensembl VEP web interface via the Ensembl navigation bar or by clicking here.

Exercises
Ex 1. We will make use of variants from the Scores file mentioned above. Download the Scores file for BAP1 exon 10 from MaveDB, and open it in any tool that can view csv files. Note the “hgvs_nt” and the “scores” columns, as we will use these for input and filtering respectively.

Ex 2. Explore the page info at BAP1 exon 10 from MaveDB.

• What does the distribution of scores look like?
• Using the “Clinical view”, do you note any pattern of Pathogenic or benign variants across score bins?
• What specific transcript ID was used for this assay?

Ex 3. Explore that transcript information by searching it on the Ensembl website. What type of transcript is this? The transcript is flagged as the MANE Select transcript of the BAP1 gene. What does the MANE Select flag tell you?

Ex 4. Use the Ensembl VEP web interface to annotate the BAP1 exon 10 variants from Exercise 1. You will need to supply the list of HGVS format variants, either by extracting that column and uploading as a file, or by copy and pasting in the list of variants. Select the following Ensembl VEP options and any others that interest you:

• enable “gnomAD (genomes) allele frequencies”
• enable “Protein Matches”
• enable “AlphaMissense” pathogenicity prediction
• enable “SpliceAI” splicing predictions
• make sure “Gene Symbol” is enabled
• make sure “MANE” is enabled

When you have enabled those options, submit the job with “Run”.

Ex 5. Once the job completes, explore your Ensembl VEP results on the web interface to answer the below. Download your results in TXT format so you can count the number of results.

• How many of these variants have been observed in the population?
• What is the most common coding consequence?
• How many results are in the output text file, why are there more than the number you submitted?

Ex 6. Use the Ensembl VEP filter options to filter your results for only those corresponding to the feature (transcript ID) to that identified in Exercise 2. Export the filtered TXT file.

Ex 7. Using the scores file from MaveDB, extract the variants with with scores lower than -0.09 only. Use this list to filter your Ensembl VEP result and explore the consequence types of these, are there any patterns that you notice? You may use the awk and grep bash commands for this step to help you, or you can try other ways of merging the input table with your Ensembl VEP results.