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Workflow 1: Viewing E. coli data in IGV
Data files
You can start this tutorial two ways:
- If you have an
intro_to_mappingdirectory with output from the Mapping tutorial or the Variant calling tutorial, then you should use those files for part 1 of this tutorial. You can proceed with either one alone or with both. - 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:
Then skip down to #Launching IGV.Code Block $BI/ngs_course/intro_to_mapping/IGV
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. So, we're going to show you another tool for sequence format conversion called Readseq.
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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! |
If you have only done the mapping tutorial and NOT the variant calling tutorial
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 in the Variant calling tutorial. If you are in your
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Repeat the last three commands for each SAM output file that you want to visualize in IGV.
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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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 on the classroom machines booted in Linux
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wget http://www.broadinstitute.org/igv/projects/downloads/IGV_2.1.22.zip unzip IGV_2.1.22.zip cd IGV_2.1.22 java -Xmx2g -jar igv.jar |
Locally on your own Mac or Windows computer
Use this link to download IGV:
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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.
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 from 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.
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
- Why are some reads different colors? Hint: Try changing the display options to show read pairs and editing some of the distance constraints.
- What is a typical mapping quality (MQ) for a read? Convert this to the probability that it is mismapped.
Expand title Remember the formula for a Phred quality score? The estimated probability that a read is mapped incorrectly is 10^(-MQ/10).
- Can you find a variant where the sequenced sample differs from the reference? This is going to be like looking for a needle in a haystack. Fortunately, we are going to learn how to use variant callers tomorrow and then we'll be able to zoom right to areas where there are discrepancies between reads and the reference genome that might indicate there were mutations in the sequenced E. coli.
Load variant calls into IGV
We're really interested in places in the genome where we think there are mutations. If you have completed the Variant calling tutorial, then you can load your VCF files to check out those spots, but first you need to (guess what?) index it.
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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.
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?
Workflow II: Viewing Human Genome Data in IGV
Advanced exercise: human data scavenger hunt
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