Hello Everyone,

I am working on a project with the goal of improving the prediction accuracy of melanoma relapse by combining the RNA signature with the clinical features.

Firstly, I calculated the asssociation/relationship between the clinical features with the outcome (early recurrence < 3yrs vs no relapse with at least 5 yrs follow up) and found that Tcat, mitosis and last_vitalstatus are closely related with the outcome result.

Secondly,I need to do the DGE by using the DESeq2 to extract a signature. During this process, I set the closely related clinical features as controls in the design to exclude their effect on the DGE result.

The R code can run successfully, but most of the generated volcano plot are weird when I consider some control factors. Only when I use the last_vitalstatus as control, the volcano plot looks normal (Fig3). The more control factors, the more stretched of the volcano plot, as presented by the last figure(VolcanoPlot_Mitoses_LastVitalStatus_Tcat_Ascontrol.jpeg). If I don't consider any control, the volcano plot is perfect, as presented by the Fig1 (VolcanoPlot_NoControl.jpeg).

The code is attached. I am very grateful, if someone can tell me if I did something wrong. As the Outcome is the feature that I am interested, so I put it at the end of the design.

dds <- DESeqDataSetFromMatrix(countData=RawCountMatrix, colData=EventControl, design= ~ mitoses+Last_vitalstatus+Tcat+Outcome_Grouping)

keep <- rowSums(counts(dds)) >= 10 dds <- dds[keep,]

dds <- DESeq(dds) res <- results(dds)

resOrdered <- res[order(res$padj),] write.csv(as.data.frame(resOrdered), file="Outcome_Good-Bad.csv")

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I set the closely related clinical features as controls in the design to exclude their effect

This can be problematic if they are highly correlated.

Compute

cor( colData(dds)[,c("factor1","factor2",...)] )

To see if they are too closely related.

Otherwise it would be helpful to show PCA plots colored by your different variables.

Hi Liuyang,

I'm just wondering whether you have managed to resolve the issue and have a normal volcano plot. I'm asking because I have ran into similar issues and all my volcano plots resemble what you got in the last one (one one big smiley face). Thank you.

Henry