I am analyzing differential gene expression for a plant based on 3'cDNA tag libraries generated from total RNA. We are looking at a fungal treatment (n=6) compared to a sterile control as well as condition (Condition W vs Condition D).
I am using DESeq2 to determine DEGs of the plant when under a fungal treatment compared to the sterile control within the condition. Then I want to use RRPP to compare DEGs and determine which DEGs are unique for each treatment and condition.
I have never used RRPP and reading up on it. Currently, when I import my count data from tximport, I set the design as follows and create a DESeq object:
ddsTxi <- DESeqDataSetFromTximport(txi, sampleTable, design = ~condition + fungal_tr + condition:fungal_tr ) keep <- rowSums(counts(ddsTxi)) > 1 ddsTxi <- ddsTxi[keep,] ddsTxi$fungal_tr <- relevel(ddsTxi$fungal_tr, ref = "CON") ddsTxi$condition <- relevel(ddsTxi$condition, ref = "W") dds_DESeq <- DESeq(ddsTxi)
So my starting questions are:
- Can I use the DESeq object and format for RRPP or do I need to transform via VST?
- Is my design incorrect for what I want to do with RRPP downstream?
Thank you and let me know what I need to clarify if need be!