Hello everyone,

I have a question related to the analysis of those genes that are differentially expressed in three mutants single KO A, single KO B and double KO C where C is a combination of mutant A + mutant B.

I am interested in analyzing the effect of combining mutant A and B to their individual effect.

So, I considered an additive effect if the double mutant is equal to the sum of the two simple mutants (δ = C - A - B = 0) and an interaction effect if δ ≠ 0.

I used the table of counts for all genes and I used DESeq2 to normalize. Then I used contrast to determine δ

This is my code:

data <- read.table("total_counts.txt",header=T,row.names=1)
groups <- factor(rep(c("CTL", "mutantA", "mutantB", "mutantC"), each=3))
sampleInfo <- data.frame(groups,row.names=colnames(data))
dds <- DESeqDataSetFromMatrix(countData = data, colData = sampleInfo, design = ~groups)
dds$groups <- relevel(dds$groups, "CTRL")
colData(dds)

DataFrame with 12 rows and 1 column
                         groups
                        <factor>
siCtrl_1                    CTRL   
siCtrl_2                    CTRL   
siCtrl_3                    CTRL   
mutantA_1               mutantA
mutantA_2               mutantA
...                         ...
mutantB_2               mutantB       
mutantB_3               mutantB       
mutantC_1               mutantC
mutantC_2               mutantC
mutantC_3               mutantC

design(dds) <- ~ 1 + groups
dds <- DESeq(dds)

estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing

resultsNames(dds)

[1] "Intercept"                     "groups_mutantA_vs_CTRL"        "groups_mutantB_vs_CTRL"        "groups_mutantC_vs_CTRL"

mod_mat <- model.matrix(design(dds), colData(dds))
mod_mat

                 (Intercept)        groupsmutantA           groupsmutantB           groupsmutantC
siCtrl_1                1                       0                       0                       0
siCtrl_2                1                       0                       0                       0
siCtrl_3                1                       0                       0                       0
mutantA_1               1                       1                       0                       0
mutantA_2               1                       1                       0                       0
mutantA_3               1                       1                       0                       0
mutantB_1               1                       0                       1                       0
mutantB_2               1                       0                       1                       0
mutantB_3               1                       0                       1                       0
mutantC_1               1                       0                       0                       1
mutantC_2               1                       0                       0                       1
mutantC_3               1                       0                       0                       1
attr(,"assign")
[1] 0 1 1 1
attr(,"contrasts")
attr(,"contrasts")$groups
[1] "contr.treatment"

A <- colMeans(mod_mat[dds$groups == "mutantA", ])
B <- colMeans(mod_mat[dds$groups == "mutantB", ])
C <- colMeans(mod_mat[dds$groups == "mutantC", ])
CTRL <- colMeans(mod_mat[dds$groups == "CTRL", ])
res <- results(dds, contrast = C - A - B)

res

log2 fold change (MLE): -1,-1,-1,+1 
Wald test p-value: -1,-1,-1,+1 
DataFrame with 58721 rows and 6 columns
                    baseMean log2FoldChange     lfcSE        stat    pvalue      padj
                   <numeric>      <numeric> <numeric>   <numeric> <numeric> <numeric>
ENSG00000000003.14  1235.661      -10.28328 0.0878839   -117.0099         0         0
ENSG00000000005.5      0.000             NA        NA          NA        NA        NA
ENSG00000000419.12  1625.580      -10.70786 0.0909223   -117.7694         0         0
ENSG00000000457.13   509.194       -8.85948 0.1080919    -81.9625         0         0
ENSG00000000460.16  1054.786       -9.75485 0.0815451   -119.6252         0         0
...                      ...            ...       ...         ...       ...       ...
ENSG00000285990.1    1.28962       -1.13755 1.8312013   -0.621204  0.534465  0.628871
ENSG00000285991.1  692.47452       -9.63571 0.0863505 -111.588309  0.000000  0.000000
ENSG00000285992.1    0.00000             NA        NA          NA        NA        NA
ENSG00000285993.1    0.00000             NA        NA          NA        NA        NA
ENSG00000285994.1    2.82204       -1.18911 1.4336056   -0.829452  0.406849  0.502778

sessionInfo( )

What I don't understand is the relationship between the log2FC and the δ. How can I determine δ from the log2FC? If I want to determine only genes with an interaction effect, or an additive effects, should I use the total table of count for all genes or should I first remove all genes where there is not significative difference between mutant A vs mutant C / mutant B vs mutant C / CTRL vs mutant C?

Thanks in advance!

Sophie

Unfortunately, I have to save my time to address software related questions. For questions about how to set up your statistical analysis, I recommend consulting with a local statistician or someone familiar with running linear models in R (DESeq2 uses the same linear model setup as base R).