PreRanked Anlysis GSEA

[Giorgia Silvestrini]

Hello everyone!! I am trying to perform a GSEA preranked analysis from DESeq2 file output. When I have to create a .rnk file, what do I have to add besides the list with the gene names? The value of log2FoldChange, stat or p-value? Which column of DESeq2 should I refer to?

Moreover, how can I understand if the KEGG pathway I get is up or downregulated? 

I happened to get a KEGG pathway with negative ES Enrichment score, but whose genes have a positive log2FoldChange so they are upregulated by expression. How can I interpret such a finding?

Thanks a lot,

Giorgia

[Anthony Castanza]

Hi Giorgia,

Depending on how many samples you have, you might be better off writing out the normalized counts table from DESeq2 and using that for standard GSEA rather than using GSEA Preranked. As for which output of the DESeq2 ranked list to use though, I would probably recommend the Log2 Fold Change column, or the Test Statistic (Stat) column, you should get reasonable (albeit slightly different) results from either of those. I would generally shy away from using the pValue, even transformed to a signed, -log10(pvalue) the correlation between the magnitude of the significance statistic and the (potential) meaningfulness of the change biologically isn't really there.

An important thing to know is that when constructing these ranked lists for input into GSEA you do not want to filter the list to just "significant" or "highly" differentially expressed genes, you'll want to use all the genes that were expressed in the samples.

Generally, we would expect that a pathway which received a negative enrichment score would be comprised mainly of genes which were downregulated. If you're seeing something different from this it would be helpful to see some screenshots at least of the results plots. Also, what did the ranked list you were using for this result look like?

-Anthony

Anthony S. Castanza, PhD

Curator, Molecular Signatures Database

Mesirov Lab, Department of Medicine

University of California, San Diego

[Giorgia Silvestrini]

Hi Anthony! 

I have done a pre-ranked analysis providing as input to the system a .rnk file containing the list of DEG genes in one column (selected using as cut-off a p-adjusted and a log2FC of my interest) and in the next column the values of log2FC. Making a comparison I got (see picture; genes refer to the cell cycle pathway) a list of pathways with negative ES and therefore downregulated, but whose genes have a positive log2FC value and therefore upregulated. 

Any suggestions?

Thank you for the help,

Giorgia

  

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[Anthony Castanza]

Hi Giorgia,

You mention that the ranked list was "selected using as cut-off a p-adjusted and a log2FC of my interest", the ranked list should not be filtered in this way. GSEA needs the entire ranked list.

Please rerun the analysis using the unfiltered ranked list and if this behavior still occurs we can evaluate what might be happening. If it does, please also include the enrichment "mountain" plot as that graph of where each gene is occurring in the global ranked list is important for debugging these kind of issues.

-Anthony

Anthony S. Castanza, PhD

Curator, Molecular Signatures Database

Mesirov Lab, Department of Medicine

University of California, San Diego

[Giorgia Silvestrini]

Got it, thanks. I went about it this way because, in reading some papers, I saw that a lot of people do this. That is, you use a list of DEGs filtered for cut-offs like you do for analysis with DAVID. However, since I have made several comparisons, only in the case that I have attached in the figure above I do not find correspondence between ES and fold change values.

Giorgia

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