2.6.3 Gene expression analysis
We identified differentially expressed transcripts (DETs) between control and Cu-treated plants from Sc3 and Sc4 using edgeR v3.24.3100. First, we filtered low count transcripts by removing those with less than 2 cpm (counts per million; i.e. less than 10 counts per transcript) in at least 3 samples to ensure that any transcript that was highly expressed in all three replicates from one group was kept. A total of 23,016 transcripts (7% of the total) passed this filter and were used in subsequent steps. Second, we fitted the model using the function glmQLFit (option robust = TRUE) and tested for DETs using the function glmTreat . Transcripts were considered differentially expressed when the FDR < 0.001 and the expression change was ≥ 4-fold (log2FC ≥ 2).
To better understand the changes associated with Cu exposure, we performed a Fisher’s exact test (with the FatiGO package101 implemented within OmicsBox) on the list of DETs from each population. With this method, we tested for significant differences in the fraction of transcripts annotated with a specific GO term between the DETs list (test set) and the full list of annotated transcripts (reference set). We applied an FDR cutoff of 0.01, and used the “Reduce to most specific ” option within OmicsBox to remove the more general, less informative, GO terms.