Hi guys, I’m working with a 8 samples experiment (lower than 3x replicates, I know..) with a design like
> colData(dds)
DataFrame with 8 rows and 2 columns
condition traitment
<factor> <factor>
4200-JS-1 norm ctrl
4200-JS-2 norm ctrl
4200-JS-3 norm trt
4200-JS-4 norm trt
4200-JS-5 hyper ctrl
4200-JS-6 hyper ctrl
4200-JS-7 hyper trt
4200-JS-8 hyper trt
I want to study the condition
effect, the traitment
effect either the differences between hyper
samples while traited. To do so I’m using this design with DESeq2 design= ~ condition + traitment + condition:traitment
. I noticed huge differences with/without using lfcShrink
while comparing either condition
and traitment
: these could be seen in the example below, where the top 3 genes (ordered by log2FC) are different and got much lower log2FC values.
Before:
log2 fold change (MLE): condition hyper vs norm
Wald test p-value: condition hyper vs norm
DataFrame with 17767 rows and 6 columns
baseMean log2FoldChange lfcSE stat pvalue
<numeric> <numeric> <numeric> <numeric> <numeric>
geneA 240.9457 21.6993 3.30544 6.56473 5.21265e-11
geneB 50.4875 21.6070 4.78560 4.51501 6.33143e-06
geneC 304.5952 21.4911 3.23791 6.63734 3.19403e-11
After:
log2 fold change (MAP): condition hyper vs norm
Wald test p-value: condition hyper vs norm
DataFrame with 17767 rows and 5 columns
baseMean log2FoldChange lfcSE pvalue padj
<numeric> <numeric> <numeric> <numeric> <numeric>
geneD 631.54567 11.96124 2.614552 6.15494e-16 1.16952e-13
geneE 21.29594 6.36406 2.750908 1.04394e-04 1.83497e-03
geneF 8.50908 4.94140 2.807262 8.57969e-04 NA
Now, in order to study the last wanted scenario, I’m using:
res=results(dds, list(c("condition_hyper_vs_norm", "conditionhyper.traitmenttrt")), alpha = 0.05)
Here I haven’t got the possibility to use lfcShrink
because of interaction term. As could be seen by the attached Volcano plot, some genes have extreme high log2FC differences as before. How to handle them? Is there any different lfcShrink
which could be used with interaction terms?
Read more here: Source link