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 close proximity.

Logging into IGV via the Interpretation Portal

Users can access IGV.js through the Interpretation Portal. For Rare Disease referrals, users can click on the genomic coordinates of the variant of interest in the variant table (Figure 1). 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 2). A set of genomic coordinates as seen in the HTML report for Cancer referrals can be seen in Figure 3 - 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) - the credentials are the same as those for the Interpretation Portal. The Login page is shown in Figure 4.

Figure 1

Figure 2

Figure 3

Figure 4

Visualising data using IGV.js

Once a user has signed in to IGV.js, they will able 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. The files for Rare Disease referrals include VCF files, coverage profiles (as BigWig files) and alignments (CRAM files). For a description of what each of these files contains, please refer to GUI-BIO-009 Rare Disease Genome Analysis Guide and GUI-BIO-010 Cancer Genome Analysis Guide.

An example showing the files available for Rare Disease referrals is shown in Figures 5.

Figure 5

After selecting files of interest, data can be visualised directly in IGV.js by pressing "Show tracks" in the top left corner (shown in Figure 6).

Figure 6

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.

To download IGV Desktop, please see the instructions here: https://software.broadinstitute.org/software/igv/download. Your local IT team will determine the appropriate download for your local computer's operating system and dependant software (i.e. Java). It is recommended to use the most up to date version of IGV.

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

For users who wish to use the desktop version of IGV, a batch script of the files for the relevant referral should be downloaded via the IGV.js page.

Users can download this batch script from the IGV.js link-out, which will enable the selected files to be loaded into IGV desktop. Clicking on the "Download batch script" button in the right hand corner of the screen will start the download which can be imported into IGV desktop.

Figure 7

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". Note that connection to OpenCGA needs to be active for the batch script to run and for files to be active once loaded in IGV Desktop.

Visualing data tracks using IGV.ja 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 8. This is denoted by the two separate read blocks annotated with the appropriate identifier (in the format ). Users can see a visualisation of the reference genome, with each colour denoting a different DNA base (adenine in green, cytosine in blue, guanine in yellow and thymine in red) with the sequencing reads for the relevant individual below. The GENCODE track shows the overlay of the gene(s) which are at the same genomic location as the variant. The dark blue track means that the transcript has been reviewed or validated by either RefSeq or SwissProt staff. Exonic regions are indicated by thick blue bars and intronic 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' beside the CRAM visualisation, as shown on the right hand side of the visualisation in Figure 8. More information on colouring alignments with a variety of variables (for example, colouring by insert size, pair orientation, both insert size and pair orientation, read strand) can be found on the 'Alignment Track' section of the 'Pop-up Menu' in the IGV User guide.

Figure 8

Visualising coverage profiles

Coverage profiles can be visualised from the appropriate BigWig files (Figure 9) with accompanying GENCODE track. These tracks show the base resolution coverage at the genomic coordinates the user is currently visualising.

Figure 9

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Last update: 2022-11-08