Hi,

I was making a contrasts using makeContrasts function for comparing groups in edgeR for differential expression analysis. I am comparing each group compared to it's baseline. Once the contrasts was created, it seems like the assignment of values (1 and -1) in the matrix is different. Ideally, Adjuvant samples should be labelled as 1 and it's respective Control samples should be -1. In the below case, it's the matrix output is different. Am I missing something or assigning differently? Perhaps, the results would be impacted due to this (especially, the direction of logFC)?

#>      Drug   Groups
#> S.1     A  Control
#> S.2     B  Control
#> S.3     C  Control
#> S.4     A  Control
#> S.5     B Adjuvant
#> S.6     C Adjuvant
#> S.7     A Adjuvant
#> S.8     B Adjuvant
#> S.9     C  Control
#> S.10    A  Control
#> S.11    B Adjuvant
#> S.12    C Adjuvant
#> S.13    A  Control
#> S.14    B  Control
#> S.15    C Adjuvant
#> S.16    A Adjuvant
#> S.17    B  Control
#> S.18    C Adjuvant

Drug.Groups <- paste(Sample_metadata$Drug, Sample_metadata$Groups, sep=".")
Drug.Groups <- factor(Drug.Groups, levels=c("B.Adjuvant", "B.Control", "C.Adjuvant", "C.Control",  "A.Adjuvant", "A.Control"))
design <- model.matrix(~0+Drug.Groups)
colnames(design) <- levels(Drug.Groups)
rownames(design) <- rownames(Sample_metadata)


Contrast.Drug.Groups <- makeContrasts(
  "A.Adj_vs_A.Con_VS_C.Adj_vs_C.Con" = (A.Adjuvant - A.Control) - (C.Adjuvant - C.Control), 
  "A.Adj_vs_A.Con_VS_B.Adj_vs_B.Con" = (A.Adjuvant - A.Control) - (B.Adjuvant - B.Control), 
  "C.Adj_vs_C.Con_VS_B.Adj_vs_B.Cont" = (C.Adjuvant - C.Control) - (B.Adjuvant - B.Control), 
  levels=design)

#>             Contrasts
#> Levels       A.Adj_vs_A.Con_VS_C.Adj_vs_C.Con A.Adj_vs_A.Con_VS_B.Adj_vs_B.Con
#>   B.Adjuvant                                0                               -1
#>   B.Control                                 0                                1
#>   C.Adjuvant                               -1                                0
#>   C.Control                                 1                                0
#>   A.Adjuvant                                1                                1
#>   A.Control                                -1                               -1
#>             Contrasts
#> Levels       C.Adj_vs_C.Con_VS_B.Adj_vs_B.Cont
#>   B.Adjuvant                                -1
#>   B.Control                                  1
#>   C.Adjuvant                                 1
#>   C.Control                                 -1
#>   A.Adjuvant                                 0
#>   A.Control                                  0

Thank you,

Mohammed

The contrast matrix is correct. It reflects exactly the interactions you defined.