Content Comparison

In this tutorial, we're going to view the aligned reads and variants that we called in the past two lessons in the Integrated Integrative Genomics Viewer from the Broad Center. You'll need the output from Introduction to mapping (bowtie, BWA) and Introduction to variant calling (SAMtools).

...

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:

• Jump right to a gene. Type its name into the search box. Try "topA".
• Navigate more quickly. The page-up page-down, home, end.
• Load multiple 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.

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

...

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.

Navigating in IGV

There are a lot of things you can do in IGV. Here are a few:

• Jump to the next point of interest. Click on a track name on the left side of the window (Ex: bowtie.vcf), to select it. You can then use control-f and control-b to 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 page-up page-down, home, end.
• 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.

...

• .

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?

...