Hello,

Thank you in advance for your help with this. I have been trying very hard to work this out and have tried to follow the vignette and looked through Q&As on this site, but to no avail.

My experimental outline: I pretreated cells to either "control" or "RecLG" for 3 days and then on the 4th day treated them to control or low glucose conditions. This gave me four groups "C", "LG", "aRLG", and "RLG".

I am interested in differential gene expression between control and RecLG pretreatments, and then the differences between each of the treatment groups. my coldata looks like this:

as.data.frame(coldata2) pretreatment treatment treatment.n AH_1 RecLG aRLG 1 AH_2 RecLG aRLG 1 AH_4 RecLG aRLG 1 AH_5 RecLG aRLG 1 AH_6 RecLG aRLG 1 CONT_1 control C 2 CONT_3 control C 2 CONT_4 control C 2 CONT_5 control C 2 CONT_6 control C 2 HYPO_1 control LG 1 HYPO_2 control LG 1 HYPO_3 control LG 1 HYPO_5 control LG 1 HYPO_6 control LG 1 RH_1 RecLG RLG 2 RH_2 RecLG RLG 2 RH_3 RecLG RLG 2 RH_5 RecLG RLG 2 RH_6 RecLG RLG 2

In each group there are 5 replicates. As you can see I made a treatment.n column to account for the nesting of "aRLG" and "RLG" within the "RecLG" pretreatment group and "C" and "LG" within the "control pretreatment group.

When I run:

dds <- DESeqDataSetFromMatrix(countData = countdata, colData = coldata2, design = ~pretreatment + pretreatment:treatment.n + treatment)

I get the error "Error in checkFullRank(modelMatrix)"

For the life of me, I can not figure this out.

The model matrix looks like this:

model.matrix(~pretreatment + pretreatment:treatment.n + treatment, coldata2) (Intercept) pretreatmentRecLG treatmentC treatmentLG treatmentRLG pretreatmentcontrol:treatment.n2 AH_1 1 1 0 0 0 0 AH_2 1 1 0 0 0 0 AH_4 1 1 0 0 0 0 AH_5 1 1 0 0 0 0 AH_6 1 1 0 0 0 0 CONT_1 1 0 1 0 0 1 CONT_3 1 0 1 0 0 1 CONT_4 1 0 1 0 0 1 CONT_5 1 0 1 0 0 1 CONT_6 1 0 1 0 0 1 HYPO_1 1 0 0 1 0 0 HYPO_2 1 0 0 1 0 0 HYPO_3 1 0 0 1 0 0 HYPO_5 1 0 0 1 0 0 HYPO_6 1 0 0 1 0 0 RH_1 1 1 0 0 1 0 RH_2 1 1 0 0 1 0 RH_3 1 1 0 0 1 0 RH_5 1 1 0 0 1 0 RH_6 1 1 0 0 1 0 pretreatmentRecLG:treatment.n2 AH_1 0 AH_2 0 AH_4 0 AH_5 0 AH_6 0 CONT_1 0 CONT_3 0 CONT_4 0 CONT_5 0 CONT_6 0 HYPO_1 0 HYPO_2 0 HYPO_3 0 HYPO_5 0 HYPO_6 0 RH_1 1 RH_2 1 RH_3 1 RH_5 1 RH_6 1 attr(,"assign") [1] 0 1 2 2 2 3 3 attr(,"contrasts") attr(,"contrasts")$pretreatment [1] "contr.treatment" attr(,"contrasts")$treatment.n [1] "contr.treatment" attr(,"contrasts")$treatment [1] "contr.treatment"

I'm just really stuck with this, I don't know where to start to sort this problem out so I've come here for help. Anything is really appreciated.

Many thanks,

Paul

FYI, session info is below: