DESeq2 Multiple Comparison Testing with Contrasts
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@jonathandiedrich-18112
Last seen 5.1 years ago

I want to run DESeq2 to identify differences in open chromatin accessibility (i.e. ATAC-seq data) between 3 different GROUPS: A, B and C. I also wanted to control for SEX and BATCH effects.

I did the analysis two ways but obtained different results and would like to understand why this is the case, and what method is more appropriate. Note that I used the same ATAC-seq counts files (i.e. the total number of sites was the same) for both analysis methods.

Method 1: Combining all groups, using LRT while running DESeq2 to control for BATCH and SEX, and then obtaining pairwise results using contrast and the Wald test afterwards

Data<-DESeqDataSetFromMatrix(countData = ABCcounts, colData = ABCinfo, design = ~BATCH+SEX+GROUP)

Data2<-DESeq(Data, test = "LRT", reduced = ~BATCH+SEX)

AvsB<-results(Data2, independentFiltering = FALSE, test = "Wald", contrast=c("GROUP","A","B"))

AvsC<-results(Data2, independentFiltering = FALSE, test = "Wald", contrast=c("GROUP","A","C"))

BvsC<-results(Data2, independentFiltering = FALSE, test = "Wald", contrast=c("GROUP","B","C"))

Method 2: Running pairwise analyses independently. Note that the counts files are identical between Method 1 and Method 2 and I only deleted columns with counts for samples in the 3rd GROUP.

DataAB<-DESeqDataSetFromMatrix(countData = ABcounts, colData = ABinfo, design = ~BATCH+SEX+GROUP)

DataAB_2<-DESeq(DataAB, test = "LRT", reduced = ~BATCH+SEX)

AB<- results(DataAB_2, independentFiltering = FALSE)

DataAC<-DESeqDataSetFromMatrix(countData = ACcounts, colData = ACinfo, design = ~BATCH+SEX+GROUP)

DataAC_2<-DESeq(DataAC, test = "LRT", reduced = ~BATCH+SEX)

AC<- results(DataAC_2, independentFiltering = FALSE)

DataBC<-DESeqDataSetFromMatrix(countData = BCcounts, colData = BCinfo, design = ~BATCH+SEX+GROUP)

DataBC_2<-DESeq(DataBC, test = "LRT", reduced = ~BATCH+SEX)

BC<- results(DataBC_2, independentFiltering = FALSE)

Can someone please explain why the results between Methods 1 and 2 are not the same? Is the first method NOT taking into account BATCH+SEX? The main goal is to look for differences between GROUPS taking into account BATCH+SEX.

Thanks!

deseq2 • 619 views
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@mikelove
Last seen 10 hours ago
United States

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