Guide to use IGV to view BAM file

[abhi_g]

Kindly provide a stepwise guide to view BAM type file on Integrative Genomics Viewer (IGV) as this is of prominent importance.
Also include details on using IGV to view BAM file result from “Samtools sort” tool.

[jennaj]

Hello @abhi_g

If you bam is not displaying yet, this means you need to configure IGV to be based on your reference genome. This can be a fasta file from the history, or a native index on the server.

Which reference genome are you currently using for mapping? Do you want to share some screenshots?

More help was in a recent topic but we can help you to navigate the instructions if we learn more about what you have now.

Let’s start here!

@jennaj

How to view data in IGV

Through the Galaxy built-in IGV viewer

1. Use the eye icon in the upper right corner of a dataset. Some data will automatically display under the Preview tab. For others, you can first click into the Visualize tab, then either 1) launch a new IGV application session 2) or join one already started up 3) or connect to your local IGV session. More about this below with a screenshot.
2. The dataset needs to represent data that is based on a reference genome assembly’s coordinates and that database key needs to be assigned. This is how the data is associated with a specific fasta index for that genome assembly.

How to troubleshoot

• Review the datatype format of the dataset. Is this coordinate based data?
• Review the database assignment. Is the data linked to a reference genome assembly? Has the dataset been annotated with this assignment?
  • If the data was generated in Galaxy using a native database key, then the key should already be assigned.
  • In some cases, you may be able to make a direct direct database assignment. This could include data processed somewhere else and Uploaded to Galaxy.
  • This works in Galaxy the same as it does when using IGV independently. Data is all based on the same assembly, and the coordinates are used to map the data to the genome’s base pairs in a fasta file. Try to avoid viewing data from different assemblies together. Why? A different assembly will use a different coordinate system. Attempting to view mixed data may result in an error or it may only have odd results in the display.

Resources

• FAQ: Changing database/build (dbkey)
• How to use IGV with a custom genome database key instead of a native genome database key. → BAM index, fasta indexes, display applications, custom genome builds, IGV - #4 by jennaj
• More about how different genome assemblies can differ, using a human assembly example. → Reference genomes at public Galaxy servers: GRCh38/hg38 example

Screenshot

This is an example for a BAM database with the native database key dm6 assigned. This dataset can be individually viewed under the Preview tab. Then, under the Visualize tab, the dataset could be viewed in a local IGV session or added to a Galaxy IGV session that includes multiple datasets all together.

If I had a custom genome key instead, then I would choose to use the local IGV session option (option 2. display with IGV – using the “local” link) – after setting up my custom genome build in Galaxy and IGV (both!). The custom build key is what informs both applications that the data is based on the same fasta index.

[abhi_g]

Thank you for the reply.
I am using hg38 as reference. After using “Samtool sort” tool on RNA STAR tool’s result, I used IGV to view the file and selected hg38 reference genome. Despite selecting the reference genome, the alignment is not displayed.

[jennaj]

Hi @abhi_g

Thanks for sharing the screenshot, very helpful!

Glad you have this all connected now! If you are not sure how to zoom into the details, try this:

Navigating IGV

1. The first pull-down menu (to the immediate right of the “IGV hg38” text), choose a chromosome (instead of “all”).
2. The second bar is free-text, and accepts coordinates.
3. You can also click around the other functions on the display to zoom/scroll/group/highlight and more including how the display is presented.

IGV has a nice user guide for learning how to navigate. This is the section where the address bar is discussed. Online videos are another resource!

IGV User Guide → https://igv.org/doc/webapp/#UserGuide/#navigating-the-view

IGV should mostly work the same in Galaxy as it does when used directly but please let us know if something seems different, or if you simply need a tip, and we can help more here!

Screenshots

Note that I converted to a bigwig to create a coverage map that can be seen at more zoom levels!

Where are the bases? Toggle that track’s option to show!

BAM datatypes

I’ll also quickly mention that a distinct step to sort a bam dataset is Galaxy is usually not needed, which may save you time, steps, and duplicated data storage. Why? All bam datasets are coordinate sorted when they are initially created and indexed as a default action.

Inspecting the bam under the Raw tab will allow you to see this notation at the top of the header in the @HD line. It will look like this.

@HD VN:1.5 SO:coordinate

This indexing occurs during Upload or when a tool produces a bam output. The datatype bam is reserved for this, so whenever that is assigned, a coordinate sort can be safely assumed and the header can confirm.

When a bam dataset is unsorted (very rare), or numerically sorted (certain protocols), the dataset will be assigned a different reserved datatype. These are based on the header content so changing the datatype yourself isn’t recommended. Instead, allow tools to interpret the data and assign the appropriate type.

Detail: bam (SO:coordinate) and qname_sorted.bam (SO:queryname) are the two bam variations most people will encounter. Use coordinate sorted bam with IGV as a qname_sorted.bam can trigger a conversion that stays in the “loading dataset” stage until it eventually fails! This would happen outside of Galaxy too!

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Hope this helps and please let us know how it goes!

[abhi_g]

Thank you very much for the guide and help.