Content Comparison

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You can start this tutorial two ways:

1. If you have ana intro_to_mapping directory with output from the Mapping tutorial or the Variant 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.

2. If you do not have any results, you can use some "canned" ones that we provide. Copy the entire contents of this directory back to your local machine:

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   /corral-repl/utexas/BioITeam/ngs$BI/gva_course/intro_to_mapping/IGV

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   Help Help title If you on on a machine with a command line...
   Code Block scp -r username@lonestar.tacc.utexas.edu:/corral-repl/utexas/BioITeam/ngsgva_course/intro_to_mapping/IGV .

   Then skip down to #Launching IGV.

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You will need to index your reference FASTA and convert your SAM output files into sorted and indexed BAM files. The "why?" behind these steps is described more fully in the Variant calling tutorial. If you are in your intro_to_mapping directory, these commands will perform the necessary steps.

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For starters, you could change into your intro_to_mapping directory and run commands like these if you just came from the Mapping the mapping tutorial:

mkdir IGV
cp NC_012967.1.fasta IGV
cp NC_012967.1.fasta.fai IGV
cp NC_012967.1.gbk.gff IGV
cp bowtie/SRR030257.sorted.bam IGV/bowtie.sorted.bam
cp bowtie/SRR030257.sorted.bam.bai IGV/bowtie.sorted.bam.bai
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wget http://www.broadinstitute.org/igv/projects/downloads/IGV_2.3.532.zip
unzip IGV_2.3.532.zip
cd IGV_2.3.532
java -Xmx2g -jar igv.jar

In a Web browser

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Go ahead and click on the "Launch with 2 GB" option. This will download a "Java Web Start" file that you can launch by locating it on your Desktop and double-clicking.

Locally on

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a Mac or Windows computer

Use this link to download IGV:

http://www.broadinstitute.org/igv/projects/downloads/IGV_2.3.532.zip

After unzipping, you should be able to click on igv.bat for Windows or igv.command on MacOSX to lauch IGV. If this is not working, you might need to try the web start.

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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 bowtie.sorted.bam

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

And you are now free to investigate different areas and their alignments in the genome.

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You can do this from within IGV:

1. Choose Tools ?  > Run igvtools....
2. Choose "index" from the commands drop-down menu.
3. Select your *.vcf file (Ex: bowtie.vcf) for "Input File"
4. Click the "run" button.

It will look like nothing has happened, but you can now close the "Run" window and choose File ? Load  > Load File. If you navigate to your IGV directory, you will now see a brand new bowtie.vcf.idx file. You can now load the file bowtie.vcf, and it will show up as a new track near the top of your window.

Tip: You can also index BAM and FASTA files the same way inside of IGV if you haven't already created indexes for them. But, it's usually easier and quicker to do this on the command line at TACC. Indexing BAM files can be a computationally hefty task. 

Exercises

• Check out the rbsA gene region? What's going on here?

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  Answer Answer
  There was a large deletion. Can you figure out the exact coordinates of the endpoints?

• Navigate to coordinate

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• 3,289,

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• 962. Compare the results for different alignment programs and settings. Can you explain what's going on here?

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  Answer Answer
  There is a 16 base deletion in the

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• gltB gene reading frame.

• What is going on in the pykF gene region? You might see red read pairs. What does that mean? Can you guess what type of mutation occurred here?

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  Answer Answer
  The read pairs are discordantly mapped. There was an insertion of a new copy of a mobile genetic element (an IS150 element) that exists at other locations in the reference sequence.

Workflow 2: Viewing Human Genome Data in IGV

If you've made it through the other exercises on your own data, take a look at some human genome re-sequencing data where the files can be loaded directly from public databases.

Advanced exercise: human data scavenger hunt 

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scavenger scavenger

See this page for the human data scavenger hunt

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1. Download and install the Integrative Genome Viewer from the Broad Institute.
2. Select "Human hg18" or "Human hg19" as the reference genome
3. Get some data: File - > Load from Server… - > 1000 genomes - > Alignments - > CEU Trio WGS - > select those 3 samples
4. Navigate to the rightmost exons of the GABBR1 gene
5. Zoom in until you find some SNPs - they might be in exons or introns; there is also at least one example of a short insertion variant between exons 2 and 3
6. Load and look at the SNP track: File - > Load from server - > Annotations - > Variants and Repeats - > dbSNP

This is whole genome coverage data; later we'll look at exome data.

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Is there an alternate allele in the child which correlates with one or both of the parents? (i.e. - do genetics work?)

From here...

You can also use IGV to visualize RNA-seq data in later tutorials.Check mapped reads and predicted variants from any later tutorial!

You may also want to check out alternative genome browsers:

• Tablet - a lightweight NGS data browser
• Visualize mapped data at UCSC genome browser