I am currently working in plant pathogen interaction and is trying to make a gene co-expression network using RNA-seq data. I used HTseq-count to get the read counts.

My 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 factor with 3 levels

condition

Day1 Day1 Day3 Day3 Day7 Day7

I am trying to get the DEGs between Day3VsDay1, Day7VsDay1 and Day7Vs_Day3. The code I used is as follows

library(DESeq2)

directory<-'D:/test_deseq/tempora'

sampleFiles<-grep('Day',list.files(directory),value=TRUE)

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'))

colData(ddsHTSeq)

ddsHTSeq <- ddsHTSeq[ rowSums(counts(ddsHTSeq)) > 1, ] ddsHTSeq dds <- DESeq(ddsHTSeq) resultsNames(dds) [1] "Intercept" "conditionDay3vsDay1" "conditionDay7vsDay1"

After running the code, to get all of the pairwise comparisons I used lfcShrink() with the contrast= option 3 times.

I tried to contrast day7VsDay3 data. But it showed an error as follows

day73 <- lfcShrink(dds, contrast=c("condition", "day7", "day3")) Error in cleanContrast(object, contrast, expanded = isExpanded, listValues = listValues, : day7 and day3 should be levels of condition such that condition_day7_vs_Day1 and condition_day3_vs_Day1 are contained in 'resultsNames(object)'

Kindly help me to resolve the error.

Is there any error in defining levels in sampleCondition andcoldata line**

how do I get all pairwise combinations of all levels espiecially Day7vsDay3 along with the other two combinations

Well, your resultsNames show that you have "conditionDay7vsDay1", not "conditionday7vsDay1"

So day73 <- lfcShrink(dds, contrast=c("condition", "Day7", "Day3"))

should work