How do I design my DESEQ experiment?
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Michael • 0
@1aa800a0
Last seen 20 months ago
Australia

I want to use DESEQ to look for differential abundant genes between my patient groups. For my samples, I have 6 patients, with each patient having 3 timepoints (baseline, infection, resolution). I want to look for DAGs with the following contrasts; Baseline vs infection, infection vs resolution.

This is what I have done so far:

dds<-DESeqDataSetFromMatrix(countData = counts, colData = metadata, design = ~ Pathology)
keep=rowSums(counts(dds)) >=10
dds=dds[keep,]
dds<-DESeq(dds)
basevsinf=results(dds, contrast = c("Pathology", "Baseline", "Infection"))

I am finding several DAGs however when I look at their individual abundance I see that they are really only present in 1 or 2 samples. I thought maybe this is an issue with my DESEQ design as I am not taking into consideration that the time points are continuous from each patient. (For example I am testing differences between the baselines of patients 1 to 6 and the infection point of patients 1 to 6). How do I include the fact that there is some within patient sampling within the design?

DESeq2 Metagenomics • 1.0k views
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swbarnes2 ★ 1.4k
@swbarnes2-14086
Last seen 13 hours ago
San Diego

You might not have enough power to detect changes in the tissues you sequenced. Adding Patient to the design might help, but might not do much.

I am finding several DAGs however when I look at their individual abundance I see that they are really only present in 1 or 2 samples.

You might want to filter those genes away, with something like

keep <- rowSums(counts(dds) >= 10) >= 3
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Thanks for the help. I did try changing the design to:

dds<-DESeqDataSetFromMatrix(countData = counts, colData = metadata, design = ~ Patient + Pathology)
keep=rowSums(counts(dds) >=10) >=3
dds=dds[keep,]
dds<-DESeq(dds)

But overall I am still seeing most of the DAGs tend to be present in only 2 samples of the 6. Is this just a short fall of only having 6 patients with 3 samples per patient?

Is there any issues with running:

keep=rowSums(counts(dds) >=10) >=4

I just tried that and I have less DAGs but they are definitely present in more samples

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Again, sometimes, you see few DE genes because she you really don't have any detectable in your samples. Do your samples group nicely in PCA? If they don't, having few DE genes would be consistent with that.

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No overall they don't group nicely so I'm not expecting many DAGs. The data looks more convincing using:

keep=rowSums(counts(dds) >=10) >=4

So I might stick that. Thanks for your help.

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