RNA-Seq Part I: Using GBrowse

There are 4 ways to browse RNA-Seq data in FlyBase and we will review them in this series of instructional videos. “RNA-Seq” refers to next-generation sequencing of the entire transcriptome for a defined developmental time, a defined
tissue, or a specific cell line. It allows detection and comparison of expression levels
of all genes active in the defined sample. We will look at an example of RNA-Seq data
using GBrowse. Navigate to a GBrowse view by going to the
gene page of your favorite gene and clicking on the GBrowse icon in the top left section of the page. Once in GBrowse, among the evidence tiers
that are open by default, you will see a colorful tier labeled “Developmental stages, unstranded
RNA-Seq”. Note that for the purpose of clarity, some default GBrowse tracks have been hidden. Mousing over the display brings up a table
of the developmental stages included in this particular analysis, arrayed top to bottom,
with a color key. In order to keep the window in view, you have to move the cursor so it
is within the window. Note that each sample is offset slightly from the prior sample,
to allow a more compact view of the data. As shown later, this display option can be
modified. Additional RNA-Seq analyses can be viewed
by clicking on the “Select Tracks” option at the top or bottom of the GBrowse page.
Scrolling down brings you to a section entitled “Expression Levels”. There are data for different
cell lines, for different treatment regimens, and for different tissue isolates. For now,
we will select two tracks and return to the GBrowse page. Return to GBrowse by clicking
“Back to Browser” at the top or bottom of the page. You can see at the top of the page the two
additional RNA-Seq datasets that were just selected. Since these are stranded data, unlike
the developmental stages previously shown, the RNA-seq view is now divided into the plus
strand and the minus strand, for each dataset. You can reposition the tier by grabbing the
label and dragging up or down. Another useful feature is formatting options
for the RNA-Seq tiers. Clicking on the icon “Configure this track” brings up a box with
several options. First, you can limit the view to a subset of the samples by selecting
adjacent samples. You can choose to increase or decrease the vertical space between the samples.
You can choose “Vertical” rather than the default “Tilted” view to remove the sample
offset. Finally, you can change the scaling method
from log2 to linear. Log2 has been chosen as the default, since it allows the best representation
of a wide range of expression, from very low to very high. A linear scale often provides
a better comparison of changes in expression for one gene across different samples.
If you click on “Apply changes” you can see the modifications that were made to the Developmental
stages tier. This tier now just shows the five selected tracks, which are vertical with
no offset and a linear scale. You may want to query the RNA-Seq data, for
example to find genes with similar expression patterns or to obtain a quantitative output for your gene of interest. Options for this type of queries will be presented in subsequent tutorials.

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