IGV | RNA-Seq Data Advanced | Alternative Splicing, Sashimi Plots

Here I’ll explain some specific skills
for studying RNA-Seq data. We’ll use human genome 18 and RNA-Seq data from liver and heart tissue samples. Let’s zoom in to get a look at the data. Here we’re looking at the expression of
a phosphate transfer gene in the heart and liver. Because we’ve loaded two datasets, we have two data tracks, one for heart RNA-Seq data, and one for liver RNA-Seq data. Each dataset gets its own coverage track and alignment track. Below the data tracks is a gene annotation track. Note that the gene annotations are
not derived from the RNA-Seq data, but have been loaded separately to help with interpretation of the RNA-Seq data. Zooming into the third and fourth exon
of this gene we see something interesting. The coverage and alignment
tracks look quite different between the tissues. Liver has high coverage of the
exon on the right whereas heart has high coverage of the exon on the left. If we expand the gene annotation track, we can see why. This phosphate transfer gene has three
different isoforms due to alternative splicing. It seems heart and liver are expressing these isoforms differently. For example, heart is expressing the third isoform significantly more than the liver. This is an important biological distinction that is easily visualized in IGV. Let’s return to our previous window using the back arrow. To better visualize the effect of splicing on the expression of this gene in heart and liver, we’re going to use a sashimi plot. You can think of a sashimi plot as a more
flexible version of the splice junction track. To open a sashimi plot, right click
and select a sashimi plot. Select the data tracks you’d like to include and click OK. In sashimi plots, the coverage for each
alignment track is plotted as bar graph. And arcs representing splice junctions
connect exons. The numbers you see on each arc are the junction depth, or how many reads span a given junction. At the bottom the gene isoforms are shown. In sashimi plots you can zoom in to see
more detail, but you can’t zoom out any farther than the zoom level of the IGV
window where you initiated the sashimi plot. By default, sashimi plots show arcs for splice junctions on the forward and
reverse strands. However, you can change this by right-clicking and selecting the
strand you’d like to see. Now for example, only the reverse strand spanning splice junctions are shown. It looks like there are none for this gene. Let’s combine the strands for now. Let’s say we don’t care about junctions
with low coverage, what we want to do is right-click and set a junction coverage minimum. When we set the junction coverage minimum to 50 we see fewer junctions, all of which have a coverage equal to or greater than 50. And if you want, you can set the junction coverage maximum in the same way. When you’re happy with your sashimi plot, you can change the color of the tracks as a final touch. And if you right-click and select save
image you can share the plot with others. Thanks for watching, be sure to check out
our other IGV tutorials.


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