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 (SamtoolsSAMtools).
Getting situated
We're going to assume that you have an introduction_to_mapping directory, and that you have existing results in those subdirectories. If not, see us and we can tell you where to copy "canned" results from.
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cd $SCRATCH/intro_to_mapping |
If you do not have the results, you can copy them using these commands. Remove any existing intro_to_mapping directory in your $SCRATCH space before running these, if you have one.
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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 .
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Prepare a GFF feature file for the reference sequence
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module load bioperl cp /corral-repl/utexas/BioITeam/ngs_course/scripts/bp_genbank2gff3.pl . ./bp_genbank2gff3.pl NC_012967.1.gbk |
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NC_012967.1.gbk.gff |
Take a look at the *.gbk and *.gff files original Genbank file and the new GFF3 file and try to get a handle on what is going on in this conversion.
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- 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.
We're going to copy all of these into a new directory called IGV to make it easier to just transfer the ones that we want.
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Now, copy this entire IGV directory back to your local 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
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.
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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 have 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 at near the top of your window.
Tip: You can index BAM and FASTA files the same way inside of IGV if you haven't already created indexes for them.
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- Jump to the next point of interest. Click on a track name on the left side of the window (Ex: bowtie.vcf), then to select it will be selected. You can then use
control-fandcontrol-bto jump forward and backward within that list of features. Try this on the variant calls track. - Jump right to a gene. Type its name into the search box. Try "topA".
- Navigate more quickly. The
homeandendkeys jump a page backward and forward. - Load multiple VCF and BAM alignments at once. Try this to compare a few different regions between the bowtie and BWA results.
- Change the appearance of genes. Right click on the gene track and try "expanded". Experiment with the other options.
- Change the appearance of reads. Right click on a BAM track and choose "show all bases" and "expanded". Experiment with the other options.
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