Here we produced a table of counts only for one mountain/locality in the interest of time. We are expanding now our analyses to 3 mountains/localities, each with the two ecotypes included. The coresponding table of counts has already been filetered as an example to retain only features/genes that are expressed over all individuals at least a number of times equal with twice the number of individuals (i.e., 36). For a scientific paper, the count tables should be filtered based on CPM (i.e., counts per million) of some value, for example 0.5 or 1 across a number of accessions equal to the smallest group in the data.

We want to run tests of differential expression for each locality separately (due to their likely independent history), so we need to include only the columns that are needed for our next analytical steps. So, use awk to prepare 3 files with counts for pair 1, for pair 4 and, respectively, for pair 5. Make use of the option -v OFS to define the Output Field Separator as tabs. For example for locality 1:

• How many individuals are included in 1counts.txt? What about how many genes?

Download the datasets in your local computer. We will continue to work locally in R for the next steps.