Overview

The Integrative Genomics Viewer (IGV) from the Broad Center allows you to view several types of data files involved in any NGS analysis that employs a reference genome, including how reads from a dataset are mapped, gene annotations, and predicted genetic variants.

Learning Objectives

In this tutorial, we're going to learn how to do the following in IGV:

• Create a custom genome database (usually used for microbial genomes) or load a pre-existing genome assembly (usually used for the genomes of model organisms and higher Eukaryotes).

• Load output from mapping reads to a reference genome.

• Load output from calling genetic variants.

• Navigate the view of the genome and interpret the display of this data.

Theory

Because NGS datasets are very large, it is often impossible or inefficient to read them entirely into a computer's memory when searching for a specific piece of data. In order to more quickly retrieve the data we are interested in analyzing or viewing, most programs have a way of treating these data files as databases. Database indexes enable one to rapidly pull specific subsets of the data from them.

The Integrative Genomics Viewer is a program for reading several types of indexed database information, including mapped reads and variant calls, and displaying them on a reference genome. It is invaluable as a tool for viewing and interpreting the "raw data" of many NGS data analysis pipelines.

Workflow 1: Viewing E. coli data in IGV

Data files

You can start this tutorial two ways:

1. If you have a mapping directory with output from the Mapping tutorial and the SNV calling tutorial, then you should use those files for part 1 of this tutorial. You can proceed with either one alone or with both.

Prepare a GFF feature file for the reference sequence

IGV likes its reference genome files in GFF (Gene Feature Format). Unfortunately, our old friend bp_seqconvert.pl doesn't deal with GFF. So, we're going to show you another tool for sequence format conversion called Readseq. We've already installed it into the $BI/bin directory so you don't have to, but here we provide the steps that can be used to install it in a local directory.

Readseq is written in java which makes it a little more complicated to use, but the general command to run the software is one of these (note that you do need to include the entire path, not just the "readseq.jar" name):

This should return the help for Readseq.

To do the conversion that we want, use this command:

It's a bit hard to figure out because, unlike most conventions, it takes the unnamed arguments before the optional flag arguments, there is no example command, and you have to switch -jar to -cp. Search online for usage examples when you can't figure something out from the help. Take a look at the contents of the original Genbank file and the new GFF file and try to get a handle on what is going on in this conversion using commands like head and tail.

Copy files to your desktop

IGV is an interactive graphical viewer program. You can't run it on TACC, so we need to get the relevant files back to your desktop machine.

They include:

• Indexed reference FASTA files

• GFF reference sequence feature files

• Sorted and indexed mapped read BAM files

• VCF result files

• ... and possibly many other types of files.

The easiest way to to this is probably to copy everything you want to transfer into a new directory called IGV_export. Since many of the tutorial output files had the same names (but resided in different directories) be careful to give them unique destination names when you copy them into the new directory together. To ensure you don't overwrite things be sure to use the -n or -i option with the cp command. The difference comes from different versions of linux having slightly different cp command options. The -n command will not allow you to overwrite files, while the -i command will prompt you before overwriting anything.

Note the need to add the suffix _fix to "samtools_tutorial" in final 4 copy steps if used the single file execution

Now, copy the entire compressed IGV directory back to your local Desktop machine.

Launching IGV

There are multiple ways to launch IGV on a local computer, in decreasing order of recommendation due to recent mac OS updates and easy of use:

Load genome into IGV

From the main window of IGV, click on Genomes > Create .genome File... and you should be presented with the following window.

Enter the ID and Name of the Genome you are working with (these can be anything that makes sense to you) and select the path to your *.fasta file (the index, *.fai file needs to be in the same directory), then select the path to your *.gff file for the Gene File. Click OK and then save this *.genome file inside the same folder as your data.

Load mapped reads into IGV

From the main window of IGV, click on File > Load from File.... Choose bowtie2.sorted.bam

After importing your reference genome and loading an alignment file, click on the + button in the upper right until reads appear! Then, your screen should look similar to the following:

Load variant calls into IGV

We're really interested in places in the genome where we think there are mutations. In the Variant calling tutorial we identified such locations but lacked a good way to visualize them. This is your opportunity to visualize them. We have already transferred the SRR030257.vcf file back to your local computer, but before we can visualize them, we need to (guess what?) index it.

You can do this from within IGV:

1. Choose Tools > Run igvtools....

2. Choose "index" from the commands drop-down menu.

3. Select the SRR030257.vcf file  for "Input File"

4. Click the "run" button.