Exercise 2: Calculating Expression Levels

In order to compare expression levels between the 2 sample conditions, you must first calculate the expression levels for each replicate. To do this, select all the assemblies produced in Exercise 1, and go to Annotate and Predict → Calculate Expression Levels. Click Continue if you get a warning that "Changes can't be undone". Use the default settings for this analysis (Ambiguously Mapped Reads: Count as partial matches, and Annotation Type: CDS) and click OK. When the analysis has finished it will automatically save, and you will get a prompt asking if you want to apply changes to the original sequences. Click Yes, then OK and then you will see an "Expression" track on each assembly.

This analysis calculates raw read, fragment and transcript counts, as well as normalized measures RPKM, FPKM and TPM. To see these values, you can either mouse over individual annotations in the Expression track, or switch to the Annotations tab above the sequence viewer to display values from the annotation track in tabular format.

A description of each expression level metric is given here.

Now switch back to the reference sequence NC_006841. Because you chose to apply the changes to the original sequence, you should be able to see 6 "Expression" annotation tracks on this sequence, one for each sample condition replicate. If you can't see them, check that they are enabled in the Annotations and Tracks tab to the right of the sequence viewer.




Now proceed to Exercise 3 to compare expression levels