In this tutorial, we're going to view the aligned reads and variants that we called in the past two lessons in the Integrated Genomics Viewer from the Broad Center. You'll need the output from Introduction to mapping (bowtie, BWA) and Introduction to variant calling (SAMtools).
Getting situated
You can start this tutorial two ways
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cds cp -r /corral-repl/utexas/BioITeam/ngs_course/intro_to_mapping/data intro_to_mapping cd intro_to_mapping cp -r /corral-repl/utexas/BioITeam/ngs_course/intro_to_mapping/samtools_* . cp -r /corral-repl/utexas/BioITeam/ngs_course/intro_to_mapping/comparison . |
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 do GFF. Fortunately, it's cousin bp_genbank2gff3.pl does.
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Another useful trick with either IGV or UCSC: displaying your own BLAST results: BioPerl allows for super-easy conversion from blast output to a gff file; IGV and the UCSC browser both understand GFF files. The short script Let's use the blast result we had from the earlier test for the JAG1 gene to show you how. You'll need to provide the input file - it's the ".oNNNNNN" output file from your blast job.
The resulting jag1_blast.out.gff can be moved to your local machine and opened in IGV. Load the human reference first though! |
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.
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In the terminal connected to Lonestar, figure out the complete path to the IGV directory.
Open a new terminal window on your Desktop. Fill in the parts in brackets <> in this command:
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Launching IGV
There are two ways; Launching IGV in your web browser or by downloading the binaries locally and running IGV from your machine.
In Browser
Navigate a web browser to this page:http://www.broadinstitute.org/software/igv/download
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
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wget http://www.broadinstitute.org/igv/projects/downloads/IGV_2.1.14.zip unzip IGV_2.1.14.zip cd IGV_2.1.14 java -Xmx2g -jar igv.jar |
Load genome into IGV
From the main window of IGV, click on File -> Import Genome and you should be presented with the following window.
Enter the ID and Name of the Genome you are working with 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.
Load mapped reads into IGV
From the main window of IGV, click on File -> Load File. Choose bowtie.sorted.bam
After importing your reference genome and loading an alignment file, your screen should look similar to the following:
And you are now free to investigate different areas and their alignments in the genome.
Load variant calls into IGV
We're really interested in places in the genome where we think there are mutations. You can load the VCF file to check out those spots, but first you need to (guess what?) index it.
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Tip: You can index BAM and FASTA files the same way inside of IGV if you haven't already created indexes for them.
Navigating in IGV
There are a lot of things you can do in IGV. Here are a few:
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See the IGV Manual for more tips and how to load other kinds of data.
Exercises
- Check out the rbsA gene region? What's going on here?
- What is going on in the pykF gene region? You might see red read pairs. What does that mean?
- Go to coordinate 2,698,092. Compare the bowtie and BWA alignments. Can you explain what's going on here?
- Go to coordinate 475,263. Compare the bowtie and BWA alignments. What's the story here?
Advanced exercise: human data scavenger hunt
Data from the CEU trio from the 1000 Genomes Project can be found directly from the Broad's server for IGV.
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