DESeq2 and heatmap for multiple comparisons
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@6e2770f3
Last seen 5 weeks ago
Germany

Hello,

I'm using DESeq2 already since some time, and I thought that I understand the procedure, but now I've stumbled upon a small problem, where I'm not sure how to proceed.

I have in my comparison 3 groups (healthy, lesional, treated), where I want to detect the genes, which are differentially expressed between healthy vs lesional, and lesional vs treated.

I'm interested in the genes which are overexpressed in lesion, but downregulated by treatment, or other way round. My problem is: I do Wald tests for 2 comparisons (healthy vs lesion, lesion vs treated) and I want to create a heatmap of differentially expressed genes (kind of time course experiment). However, if I take dds results with reference="healthy", then everything is compared to "healthy" cohort, and the data in the heatmap also displays comparison to ''healthy". What would be the best way of doing that? Theoretically, if I would take "lesion" as reference, it could solve the problem, but it would display inverse correlations...

I thought of extracting assay data from these 2 Wald tests for the genes, overlapping between 2 groups, then merge the data and create Heatmap. In this case, should I take raw counts and then perform normalization, or take already normalized values? Or is LRT the better way? I suppose it has already been discussed once, but I could not find an answer to that question.

Thank you

DESeq2 • 328 views
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Sorry, I think it was my mistake - false understanding of the produced heatmap.

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swbarnes2 ★ 1.2k
@swbarnes2-14086
Last seen 3 hours ago
San Diego

If you want to know what genes are different between lesion and treated, compare them to each other.

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My question is how to visualize differentially expressed genes on the heatmap for all 3 groups (healty->lesion->treated) if in the first analysis "healthy" is the reference, and in the second analysis "lesion". I want to graphically demonstrate the genes up- or downregulated by inflammation, which are then modified by treatment. I don't want to use 2 different volcano plots. Heatmap using "lesion" as reference for DESeq gives nice results, but I would like to demonstrate changes in proper direction, and not healthy<-lesion->treated, but I'm not quite sure how to get to this data.

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@6e2770f3
Last seen 5 weeks ago
Germany

I finally understood how I can visualize heatmap with 3 groups simultaneously, even if it's not the recommended method. I extracted raw counts from assay(dds), then performed variance stabilizing transformation and extracted only the data for differentially expressed genes of interest. After scaling the data into z-scores, I could produce heatmap, demonstrating differences between all 3 groups.

However, as far as I know, usage of raw counts is not a proper way, but I did not know how to solve this problem in other way.