Hi,

I am working with multi-factor RNA-Seq experiment in EdgeR to find diff. expressed genes. The samples were processed in different batches (see below). I am trying to compare two different effects of Treatment A, and B paired samples. Additionally, I am also interested in comparisons of two groups Group_1 and Group_2, though the main goal is to compare between Treatment B vs. A.. I have a question about creating a right design matrix for the comparisonsa and use it in estimateDisp and glmQLFit.

g.Treatments <-  factor(Samples$Treatments)
g.Groups <-  factor(Samples$Groups)
g.Batch <-  factor(Samples$Batch)
y <- DGEList(counts = data_counts, group = group.Treatments, remove.zeros = TRUE)

I am interested in comparing 2 Treatment, and find diff exp genes:

    des_treat.1 <- model.matrix(~Batch+Individuals+Treatments) 
(OR)
    des_treat.2 <- model.matrix(~Individuals+Batch+Treatments)

I am interested in comparing 2 groups, and find diff exp genes:

    des_group.1 <- model.matrix(~Batch+Groups)
(OR)
    des_group.1 <- model.matrix(~Groups)

OR, use all comparison in same line, is it possible?

des_treat.group.1 <- model.matrix(~Batch+Individual+Treatments+Groups)
des_treat.group.2 <- model.matrix(~Individual+Batch+Treatments+Groups)

Samples

#>       Individuals Treatments  Groups Batch
#> ID_1            1          A Group_1     1
#> ID_2            1          B Group_1     1
#> ID_3            2          A Group_2     2
#> ID_4            2          B Group_2     2
#> ID_5            3          A Group_1     1
#> ID_6            3          B Group_1     1
#> ID_7            4          A Group_2     2
#> ID_8            4          B Group_2     2
#> ID_9            5          A Group_1     1
#> ID_10           5          B Group_1     1
#> ID_11           6          A Group_2     2
#> ID_12           6          B Group_2     2

Thank you,

Abdul

See Section 3.5 "Comparisons both between and within subjects" in the edgeR User's Guide.

No, you cannot include a batch effect because your batches are completely confounded with the two groups of individuals. The analysis described in the edgeR Users Guide does not make baseline comparisons between the batches and hence is unaffected by any batch effect. You need to follow the edgeR analysis as it is.