ssGSEA RNAseq input expression data

[jheey1521]

I have raw read count values generated by HTSeq-count and normalized counts generated by DESeq2 and FPKM values.

I know that I need an input GCT file containing normalized gene expression data to run ssGSEA.

And I confirmed from the GSEA website that normalized counts generated by DESeq2 can be used as input GCT files for GSEA analysis.

"Tools such as DESeq2 can be made to produce properly normalized data (normalized counts) which are compatible with GSEA"

Then, could the normalized counts created by DESeq2 be used as gene expression data to be put into the input GCT file for ssGSEA analysis?

Thank you in advance.  

[Anthony Castanza]

Hello,

For ssGSEA we recommend TPM (transcripts per million), or FPKM (fragments per kilobase million). 

Counts or normalized counts are not suitable for ssGSEA as the value is not normalized for the length of the gene. This results in a bias for longer genes that wouldn't be accounted for when computing relative enrichment of gene sets within the single sample.  This doesn't affect standard GSEA as the gene ranking is computed relative to other samples.

-Anthony

Anthony S. Castanza, PhD
Curator, Molecular Signatures Database
Mesirov Lab, Department of Medicine
University of California, San Diego

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