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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:
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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
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You can do this from within IGV:
- Choose Tools ? > Run igvtools....
- Choose "index" from the commands drop-down menu.
- Select your *.vcf file (Ex:
bowtie.vcf) for "Input File" - Click the "run" button.
It will look like nothing has happened, but you can now close the "Run" window and choose File ? > 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?
Expand Answer Answer There was a large deletion. Can you figure out the exact coordinates of the endpoints?
Navigate to coordinate 4753,289,288962. Compare the results for different alignment programs and settings. Can you explain what's going on here?
Expand Answer Answer There is a 16 base deletion in the gltA 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?
Expand 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.
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