Biological replicates in DESeq2
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Blanca • 0
@blanca-24929
Last seen 6 months ago

Hi, This is my first time using the DESeq2 package for RNA-seq analysis. I am analyzing a study of three different time points in mice (5,10 and 15 days old), all mice with the same genotype, and 3 biological replicates per age. I want to evaluate differential expression as an effect of the different ages. I am confused on how I should correct for the variability associated to the biological replicates when checking for the effect of age.

I have tried both these options for the code but I always get very small p-values (mostly 0) and very high number of genes differentially expressed (half of the detected genes), which I don't think makes any biological sense.  Option 1:

Option 2:

# design= ~condition + rep2),

Any help with how to properly use "design" would be greatly appreciated!

DESeq2 Bioconductor RNASeqR • 225 views
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ATpoint ▴ 840
@atpoint-13662
Last seen 19 minutes ago
Germany

For you it would simply be:

DESeqDataSetFromMatrix(countData=countdata,
colData=data.frame(condition = factor(rep(c("P10", "P15", "P5"), each=3))),
design="~condition")


Each element of colData with the same factor is considered a replicate, there is no need for (it would even be wrong) something like ~rep`.

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