My RNA-seq dataset consist of Resis_treated (1st day, 3rd day and 7th day) samples taken under 3 time point with 2 replicated for each time point. I have a condition with 3 levels (day1, day3, day7). I used HTseq-count to get the read counts.
I am trying to get the DEGs between Day3VsDay1, Day7VsDay1 and Day7Vs_Day3. After running the code, to get all of the pairwise comparisons I used lfcShrink() with the contrast= option 3 times.
sampleCondition<-c('Day1','Day1','Day3','Day3', 'Day7','Day7') sampleTable<-data.frame(sampleName=sampleFiles, fileName=sampleFiles, condition=sampleCondition) ddsHTSeq<-DESeqDataSetFromHTSeqCount(sampleTable=sampleTable, directory=directory, design=~condition) colData(ddsHTSeq)$condition<-factor(colData(ddsHTSeq)$condition, levels=c('Day1','Day3','Day7')) ddsHTSeq <- ddsHTSeq[ rowSums(counts(ddsHTSeq)) > 1, ] dds <- DESeq(ddsHTSeq)
After getting all the three combinations
`day73 <- lfcShrink(dds, contrast=c("condition", "day7", "day3")) day71 <- lfcShrink(dds, contrast=c("condition", "day7", "day1")) day31 <- lfcShrink(dds, contrast=c("condition", "day3", "day1"))`
In the result table of all the three combinations saved separately, It was observed that the base mean value was same for all of three combinations
And also the log2fc is very less in each combination when compared to the results of all the wald test done for each combination (Day3VsDay1, Day7VsDay1 and Day7Vs_Day3) individually as separate three DESeq run. Why is it so?