I am attempting to use GSEA enrichr with 10x genomics differential_expression rankings. Reading what people seem to be doing with GSEA, there seems to be a pre-/post-singlecell gene expression versioning of the tools associated, so I want to make sure what I am doing makes sense.
For each gene in columns 1 and 2, there are 3 columns in each of the clusters that the 10X Genomics
cellranger count software produces, e.g.:
Cluster 3 Mean Counts Cluster 3 Log2 fold change Cluster 3 Adjusted p value 0.2392551160479023 -6.591931364165717 0.029177998608334674 0.1435530696287414 -5.608385347948343 0.0003833420249831818 1.107409394278862 -5.441829814465051 3.8059567638748296e-09 0.1948220230675776 -4.9155704967404 0.008739337761029243 0.20849374398460058 -4.65702722396748 1.0980978990257375e-11 0.36571853453036496 -4.6498340596466585 1.1896434946397147e-17 0.1811503021505546 -4.445327449243366 1.3416233207430882e-12 0.4409129995739914 -4.433552335619653 1.2400396349891802e-16 1.7226368355448967 -4.240367394494225 2.2072280062348652e-15 0.10937376733618391 -3.902215199856403 2.0315030904651526e-12 3.2948847410025404 -3.7580328996321235 0.028858857004902847 0.3588826740718535 -3.738237134795285 8.439629283346646e-13 [...]
In trying to give a tissue / cell type identity to each of these clusters, I've used the following method to feed a subset of genes to EnrichR:
1- Sort the
Log Fold change column numerically so we get low negative values.
2- Filter out for the
Adjusted p-value to have a value smaller than 0.05.
3- Filter out for the
Means Counts value to be bigger than 0.01.
4- Take the top n genes from the list, and feed it to GSEA EnrichR to compare against Mouse_Gene_Atlas or ARCHS4_Tissues.
Given that the fold change of the clusters is relative to each other, does it make more sense to rank the top n genes numerically or in inverse numerical value? E.g. selecting the first 100 genes in each cluster that have the most negative
Log2 fold change values or the most positive
Log2 fold change values or both (the top 100 genes with highest absolute
Log2 fold change)?
Looking at previous answers, I get that one shouldn't rank by p-value: e.g. see @gringer reply https://bioinformatics.stackexchange.com/a/13465/180