How to Visualize bam files & find mutations

1. Launch the IGV (integrated genomics viewer) maintained by the broad institute.
2. **Switch from the human hg18 reference genome to the human hg19 reference genome. The image below shows the drop down menu in the top left of the IGV
3. Go to File, choose Load from file and select the file Stacy's FGD4 gene (included are the FGD4.bam and FGD4.bam.bai files required for visiualization).
4. Type in FGD4 and press ENTER, as shown below...

This gene is large and you may have to zoom in to examine it up close.

In the same text box where the gene FGD4 was entered, type in the following region:

chr12:32,778,799-32,778,882 to zoom into a smaller and more visible region

You should now see something like this...

Each gray region in the middle window represents a position in the exome that matches the reference (consensus)  sequence. Each contiguous gray "chunk" is called a read. It's an attempt to read a small section of the genome. If there is a gray region in the read, chances are it's not causing any diseases since it's seen the majority of the time. Sometimes faulty reads will show a mutation that is not really there. A real mutation is likely to show multiple reads with the mutation. This window shows red and blue colors representing T and C alleles, respectively. The gray regions in between the two vertical dashed lines are C alleles. You can right-click the box containing the text FGD4.bam and select "show all bases" in order to visualize every single base pair read. I prefer to leave the "show all bases" unchecked so that any abnormal regions will stand out as regions that match will reference will be colored gray and can be ignored. The T's show up as red to emphasize that they do not match the reference sequence, meaning this is a mutation that is not seen a majority of the time. This particular T allele is in an intron within the FGD4 gene, which is why the bottom horizontal line simply shows blue arrows that show the direction in which the gene is expressed (more on that later).

In the next section, we'll check Stacy's FGD4 gene for specific mutations known to cause CMT4.