Using DESeq with 2 assays having 2 conditions
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@63286cde
Last seen 3.3 years ago

Hi everyone,

I have two assays : fraction and cell (for cell fraction and whole cell experiments, which have been made separately)

and two conditions tested in those two assays: treatment and control.

I don't know if it's relevant here, but I have 3 replicates per conditions, so a total of 12.

I followed this post , because I want to see the DE in the fraction when it's treated compared to the cell, but also compared to control (fraction and cell). We decided to take this design then:

(fraction_treated / cell_treated) / (fraction_control / cell_control)

So my colData have 2 columns : assay and condition

I then used this code (found in the post cited above):

dds <- DESeqDataSetFromMatrix(countData = countData,
                              colData = colData,
                              design = ~ assay + condition + assay:condition)
dds <- DESeq(dds, test="LRT", reduced= ~ assay + condition)
results(dds)

I am not sure if I am doing something wrong, because when I use resultsNames(dds), I obtain this:

#[1] "Intercept"                "assay_Fraction_vs_Cell"         "condition_treatment_vs_control" "assayFraction.conditionTreatment"

If it's the way it should be, does the results will correspond to my design? Or maybe should I use a different contrast?

DESeq2 • 738 views
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swbarnes2 ★ 1.3k
@swbarnes2-14086
Last seen 1 day ago
San Diego

Given what you want to do, to get that ratio of ratios, your design is correct. I'm not sure you need to do the LRT part, that's usually for when you have a whole bunch of different conditions, and you want to find something that is different between any two of those conditions, without having to call 10 different contrasts.

The "assayFraction.condition.control" contrast is what you want, though you might want to relevel to make sure that the references for treatment and condition are what you want.

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Thank you very much for your answer! By the way, I made a mistake in my last output, it was "assayFraction.conditionTreatment" and not "assayFraction.conditionControl". I corrected it.

So here is what I did:

dds <- DESeq(dds)
dds$condition<- relevel(dds$condition, "treatment")
colData(dds)

Gives me this:

                       condition     assay        sizeFactor
                       <factor>      <factor>     <numeric>
repA_1_Cell             control       Cell         2.61397
repA_1_Fraction         control       Fraction     1.00655
repA_2_Cell             control       Cell         2.44332
repA_2_Fraction         control       Fraction     1.09813
repA_3_Cell             control       Cell         1.90305
...                      ...         ...            ...
repB_1_Fraction         treatment     Fraction     0.264316
repB_2_Cell             treatment     Cell         2.166999
repB_2_Fraction         treatment     Fraction     0.218834
repB_3_Cell             treatment     Cell         1.767073
repB_3_Fraction         treatment     Fraction     0.210361

And finally:

res <- results(dds, name="assayFraction.conditionTreatment")

I made a volcano plot and a MA plot out if this, but I have to say, I am a little bit disappointed, because there are not that much DE genes. Do you see a mistake here or do you think everything is ok? Thanks again!

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Maybe your treatments and conditions don't have much an effect. Does your PCA show clear separation?

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There is a clear separation between conditions in the fractions, but not that much for Cell. I think it's normal, because the treatment is not suppose to have a big effect on the cell.

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