Using IGV

Background

Visualising small variants (single nucleotide variants (SNV) and indels), copy number variants (CNVs) and other structural variants (SVs) can be helpful for numerous reasons, including but not limited to the following:

1. Assessing variant quality, e.g. to determine whether a variant is likely to be a true variant rather than a sequencing error or artefact.
2. Assessing a variant in context with other nearby variants, e.g. to determine whether a selected variant is part of a more complex event.
3. Assessing a variant or region of interest alongside coverage data, e.g. to determine whether an interval has adequate sequencing depth.
4. Identifying, assessing and interpreting breakpoints for structural variants.
5. Confirming inheritance patterns for variants of interest in Rare Disease families, e.g. paternal, maternal, both, or de novo. Assessing phase of variants in proximity.

Logging in to IGV

Logging in to IGV for Rare Disease referrals

Users can access IGV through the Interpretation Portal.

For Rare Disease referrals, users can click on the genomic coordinates of the variant of interest in the variant table of the Interpretation Browser (Figure 1).

Rare disease referrals can also be examined in IGV via the New Interpretation Browser (New IB).

On the New IB's Small Variant Grid clicking on the IGV symbol next to a variant's genomic coordinate will bring the user to IGV (Figure 2). The New IB also links to IGV from the variant details page via a link in the top right corner.

Logging in to IGV for Cancer referrals

For Cancer referrals, users can access IGV.js via links presented in the HTML Report, which can be downloaded from the "Associated Files" section of the Cancer referral main page in the Interpretation portal (Figure 3).

A set of genomic coordinates as seen in the HTML report for Cancer referrals can be seen in Figure 4 - these coordinates link out to IGV, as those in the Rare Disease variant table do.

Clicking on a coordinate hyperlink will take users to the login page for IGV.js (via OpenCGA).

IGV can also be accessed from the Cancer Decision Support System (CDSS). On The CDSS variant list, clicking on the IGV logo next to a variant's genomic coordinates will load IGV for that variant. IGV can also be accessed for a given variant from the expanded 'variant details' page.

The IGV login page (for both Rare Disease and Cancer) is shown in Figure 5.

Credentials and network

Your login credentials for IGV should be the same as those for the Interpretation Portal.

To access IGV you must be connected to the HSCN network.

If you experience issues logging in please contact the Genomics England Service Desk.

Figure 1: Clicking on the variant coordinates or rsid (shown in the red box) will bring the user to the IGV web application.

Figure 2: On the New IB, click the IGV icon next to variant coordinates to access IGV.

Figure 3: For cancer cases, users can access IGV by downloading the cancer report HTML files found in the 'Associated Files' section of the IP and following the variant links found within.

Figure 4: From within a downloaded cancer report, the link to link-out to IGV for a given variant appears as shown in the red box.

Figure 5: The IGV login page.

Visualising data using the IGV.js web application

Once a user has signed in to IGV.js, they will be able to see a list of files associated with the selected referral. These files can be visualised in IGV.js or in IGV desktop using the batch script.

A description of the files available to view in IGV is available in the Rare Disease Genome Analysis guide and the Cancer Genome Analysis guide.

The files for Rare Disease referrals include VCF files, coverage profiles (as BigWig files) and alignments (CRAM files), which are commonly visualised using IGV.

An example showing the files available for Rare Disease referrals is shown in Figure 6.

Figure 6: An example of available files for a rare disease case.

After selecting files of interest, data can be visualised directly in IGV.js by pressing "Show tracks" in the top left corner (Figure 7).

Figure 7: Clicking 'Show tracks' as highlighted in the red box will display the selected files.

Using IGV Desktop

Users may prefer to perform data visualisation on their local computer using IGV Desktop (where local IT systems allow).

Downloading IGV Desktop may also produce better loading times than the web-based IGV.js, and provides a more extensive set of features. IGV Desktop is recommended for visualising structural and copy number variants.

To download IGV Desktop, please see the instructions here: https://software.broadinstitute.org/software/igv/download.

It is recommended to use the most up-to-date version of IGV.

Downloading IGV Desktop

Genomics England do not develop IGV.

When installing IGV on your local system please ensure you follow your local IT guidance.

If you experience issues with the installation of IGV Desktop please contact your local IT team, or try troubleshooting on the developer's webpage.

Downloading batch files from the Interpretation Portal for visualisation in IGV desktop

For users who wish to use the desktop version of IGV, the selected data files can be viewed by running a batch script in IGV.

To load files in IGV desktop, a batch script of the files for the relevant referral should be downloaded via the IGV.js page. After selecting the files of interest, users should click on the "Download batch script" button in the right hand corner of the screen. This will download the relevant batch script which can be imported into IGV desktop.

Figure 8: Clicking 'Download batch script', as shown in the red box, will download a script to be run in IGV desktop to visualise the selected files.

Running the batch script in IGV Desktop

In the IGV desktop application, users can run the batch script they have downloaded by opening the IGV desktop application and clicking on the "Tools" button at the top of the window.

This will open a drop-down menu, from which users should select "Run Batch Script", choose the file they have downloaded, and then press "Open".

Figure 9: In IGV Desktop, select 'Tools' and then 'Run Batch Script' to visualise downloaded data.

IGV Desktop connection issues

Users must remain connected to HSCN to visualise GMS data in IGV Desktop using the batch script! If the connection is closed, or the connection with IGV.js times out, a new batch script must be downloaded.

The following video shows you how to download and run batch scripts in IGV Desktop

Visualising data tracks using IGV.js or IGV desktop

Visualising genome alignments

Genome alignments can be visualised from the appropriate CRAM files. An example of CRAM visualisation for a rare disease referral with two patients can be seen in Figure 10. This visualisation is denoted by the two separate read blocks annotated with identifiers shown on the top left of each red blocks. Identifiers for cram files are formatted as [Sample ID]-[Plate Well].cram.

Users can see a visualisation of the reference genome, with each colour denoting a different DNA base. The sequencing reads for the relevant individuals are shown below the reference genome. The GENCODE track at the bottom of the screen shows the gene(s) which are in the same genomic location as, or in the vicinity of, the variant. In the GENCODE tracks, exons are indicated by thick blue bars and introns regions by thinner blue lines.

The end of the read will show as a blunt end or a pointed end, with the pointed end denoting the directionality of the alignment. Alignments can be coloured by read strand by clicking on the 'cog' symbol to the upper right of the CRAM visualisation (Figure 10). Alignment reads can also be coloured by a variety of variables (for example, colouring by insert size, pair orientation, both insert size and pair orientation, or read strand).

Full IGV Documentation

The full IGV documentation with more comprehensive instructions for data visualisation can be found here on the developers' webpage

Figure 11: A CRAM file visualised in IGV.

Visualising coverage profiles

Coverage profiles can be visualised from the appropriate BigWig files (Figure 11) with accompanying GENCODE track. These tracks show the base resolution coverage at the genomic coordinates the user is currently visualising.

Figure 11: Coverage profiles from a BigWig file visualised in IGV.