Using fgsea package

Loading necessary libraries

library(fgsea)
library(data.table)
library(ggplot2)

Quick run

Loading example pathways and gene-level statistics and setting random seed:

data(examplePathways)
data(exampleRanks)
set.seed(42)

Running fgsea:

fgseaRes <- fgsea(pathways = examplePathways, 
                  stats    = exampleRanks,
                  minSize  = 15,
                  maxSize  = 500)

The resulting table contains enrichment scores and p-values:

head(fgseaRes[order(pval), ])

##                                            pathway         pval         padj
##                                             <char>        <num>        <num>
## 1:                     5990979_Cell_Cycle,_Mitotic 6.690481e-27 3.920622e-24
## 2:                              5990980_Cell_Cycle 3.312565e-26 9.705816e-24
## 3:                    5991851_Mitotic_Prometaphase 8.470173e-19 1.654507e-16
## 4: 5992217_Resolution_of_Sister_Chromatid_Cohesion 2.176649e-18 3.188791e-16
## 5:                                 5991454_M_Phase 1.873997e-14 2.196325e-12
## 6:         5991599_Separation_of_Sister_Chromatids 8.733223e-14 8.529448e-12
##      log2err        ES      NES  size                              leadingEdge
##        <num>     <num>    <num> <int>                                   <list>
## 1: 1.3422338 0.5594755 2.769070   317 66336,66977,12442,107995,66442,12571,...
## 2: 1.3267161 0.5388497 2.705894   369 66336,66977,12442,107995,66442,19361,...
## 3: 1.1239150 0.7253270 2.972690    82 66336,66977,12442,107995,66442,52276,...
## 4: 1.1053366 0.7347987 2.957518    74 66336,66977,12442,107995,66442,52276,...
## 5: 0.9759947 0.5576247 2.554076   173 66336,66977,12442,107995,66442,52276,...
## 6: 0.9545416 0.6164600 2.670030   116 66336,66977,107995,66442,52276,67629,...

As you can see from the warning, fgsea has a default lower bound eps=1e-10 for estimating P-values. If you need to estimate P-value more accurately, you can set the eps argument to zero in the fgsea function.

fgseaRes <- fgsea(pathways = examplePathways, 
                  stats    = exampleRanks,
                  eps      = 0.0,
                  minSize  = 15,
                  maxSize  = 500)

head(fgseaRes[order(pval), ])

##                                            pathway         pval         padj
##                                             <char>        <num>        <num>
## 1:                              5990980_Cell_Cycle 2.535645e-26 1.485888e-23
## 2:                     5990979_Cell_Cycle,_Mitotic 9.351994e-26 2.740134e-23
## 3:                    5991851_Mitotic_Prometaphase 3.633805e-19 7.098033e-17
## 4: 5992217_Resolution_of_Sister_Chromatid_Cohesion 2.077985e-17 3.044248e-15
## 5:                                 5991454_M_Phase 2.251818e-14 2.639131e-12
## 6:          5991502_Mitotic_Metaphase_and_Anaphase 3.196758e-14 3.122167e-12
##      log2err        ES      NES  size                              leadingEdge
##        <num>     <num>    <num> <int>                                   <list>
## 1: 1.3344975 0.5388497 2.664606   369 66336,66977,12442,107995,66442,19361,...
## 2: 1.3188888 0.5594755 2.740246   317 66336,66977,12442,107995,66442,12571,...
## 3: 1.1330899 0.7253270 2.926512    82 66336,66977,12442,107995,66442,52276,...
## 4: 1.0768682 0.7347987 2.920436    74 66336,66977,12442,107995,66442,52276,...
## 5: 0.9759947 0.5576247 2.547515   173 66336,66977,12442,107995,66442,52276,...
## 6: 0.9653278 0.6052907 2.639370   123 66336,66977,107995,66442,52276,67629,...

One can make an enrichment plot for a pathway:

plotEnrichment(examplePathways[["5991130_Programmed_Cell_Death"]],
               exampleRanks) + labs(title="Programmed Cell Death")

Or make a table plot for a bunch of selected pathways:

topPathwaysUp <- fgseaRes[ES > 0][head(order(pval), n=10), pathway]
topPathwaysDown <- fgseaRes[ES < 0][head(order(pval), n=10), pathway]
topPathways <- c(topPathwaysUp, rev(topPathwaysDown))
plotGseaTable(examplePathways[topPathways], exampleRanks, fgseaRes, 
              gseaParam=0.5)

From the plot above one can see that there are very similar pathways in the table (for example 5991502_Mitotic_Metaphase_and_Anaphase and 5991600_Mitotic_Anaphase). To select only independent pathways one can use collapsePathways function:

collapsedPathways <- collapsePathways(fgseaRes[order(pval)][padj < 0.01], 
                                      examplePathways, exampleRanks)
mainPathways <- fgseaRes[pathway %in% collapsedPathways$mainPathways][
                         order(-NES), pathway]
plotGseaTable(examplePathways[mainPathways], exampleRanks, fgseaRes, 
              gseaParam = 0.5)

To save the results in a text format data:table::fwrite function can be used:

fwrite(fgseaRes, file="fgseaRes.txt", sep="\t", sep2=c("", " ", ""))

To make leading edge more human-readable it can be converted using mapIdsList (similar to AnnotationDbi::mapIds) function and a corresponding database (here org.Mm.eg.db for mouse):

library(org.Mm.eg.db)
fgseaResMain <- fgseaRes[match(mainPathways, pathway)]
fgseaResMain[, leadingEdge := mapIdsList(
                                     x=org.Mm.eg.db, 
                                     keys=leadingEdge,
                                     keytype="ENTREZID", 
                                     column="SYMBOL")]
fwrite(fgseaResMain, file="fgseaResMain.txt", sep="\t", sep2=c("", " ", ""))

Performance considerations

Also, fgsea is parallelized using BiocParallel package. By default the first registered backend returned by bpparam() is used. To tweak the parallelization one can either specify BPPARAM parameter used for bplapply of set nproc parameter, which is a shorthand for setting BPPARAM=MulticoreParam(workers = nproc).

Using Reactome pathways

For convenience there is reactomePathways function that obtains pathways from Reactome for given set of genes. Package reactome.db is required to be installed.

pathways <- reactomePathways(names(exampleRanks))
fgseaRes <- fgsea(pathways, exampleRanks, maxSize=500)
head(fgseaRes)

##                                                            pathway       pval
##                                                             <char>      <num>
## 1:                      5-Phosphoribose 1-diphosphate biosynthesis 0.88050314
## 2: A tetrasaccharide linker sequence is required for GAG synthesis 0.49554367
## 3:                           ABC transporters in lipid homeostasis 0.18160920
## 4:                          ABC-family proteins mediated transport 0.40845070
## 5:                                    ABO blood group biosynthesis 0.97703549
## 6:                       ADP signalling through P2Y purinoceptor 1 0.01208293
##         padj    log2err         ES        NES  size  leadingEdge
##        <num>      <num>      <num>      <num> <int>       <list>
## 1: 0.9526383 0.05367696  0.4267378  0.6746132     2 328099, ....
## 2: 0.7756544 0.07362127  0.3755168  0.9660172    10 14733, 2....
## 3: 0.5140597 0.15631240 -0.4385385 -1.2574076    12 19299, 2....
## 4: 0.7328846 0.07687367  0.2614189  1.0268252    66 17463, 2....
## 5: 0.9911908 0.04870109  0.5120427  0.6902345     1        14344
## 6: 0.1037197 0.38073040  0.6097588  1.7826019    17 14696, 1....

Starting from files

One can also start from .rnk and .gmt files as in original GSEA:

rnk.file <- system.file("extdata", "naive.vs.th1.rnk", package="fgsea")
gmt.file <- system.file("extdata", "mouse.reactome.gmt", package="fgsea")

Loading ranks:

ranks <- read.table(rnk.file,
                    header=TRUE, colClasses = c("character", "numeric"))
ranks <- setNames(ranks$t, ranks$ID)
str(ranks)

##  Named num [1:12000] -63.3 -49.7 -43.6 -41.5 -33.3 ...
##  - attr(*, "names")= chr [1:12000] "170942" "109711" "18124" "12775" ...

Loading pathways:

pathways <- gmtPathways(gmt.file)
str(head(pathways))

## List of 6
##  $ 1221633_Meiotic_Synapsis                                                : chr [1:64] "12189" "13006" "15077" "15078" ...
##  $ 1368092_Rora_activates_gene_expression                                  : chr [1:9] "11865" "12753" "12894" "18143" ...
##  $ 1368110_Bmal1:Clock,Npas2_activates_circadian_gene_expression           : chr [1:16] "11865" "11998" "12753" "12952" ...
##  $ 1445146_Translocation_of_Glut4_to_the_Plasma_Membrane                   : chr [1:55] "11461" "11465" "11651" "11652" ...
##  $ 186574_Endocrine-committed_Ngn3+_progenitor_cells                       : chr [1:4] "18012" "18088" "18506" "53626"
##  $ 186589_Late_stage_branching_morphogenesis_pancreatic_bud_precursor_cells: chr [1:4] "11925" "15205" "21410" "246086"

And running fgsea:

fgseaRes <- fgsea(pathways, ranks, minSize=15, maxSize=500)
head(fgseaRes)

##                                                                                    pathway
##                                                                                     <char>
## 1:                                                                1221633_Meiotic_Synapsis
## 2:                                   1445146_Translocation_of_Glut4_to_the_Plasma_Membrane
## 3: 442533_Transcriptional_Regulation_of_Adipocyte_Differentiation_in_3T3-L1_Pre-adipocytes
## 4:                                                                  508751_Circadian_Clock
## 5:                                               5334727_Mus_musculus_biological_processes
## 6:                                        573389_NoRC_negatively_regulates_rRNA_expression
##         pval      padj    log2err         ES        NES  size  leadingEdge
##        <num>     <num>      <num>      <num>      <num> <int>       <list>
## 1: 0.5483333 0.7251271 0.06523531  0.2885754  0.9412385    27 15270, 1....
## 2: 0.6857610 0.8336764 0.05378728  0.2387284  0.8387184    39 17918, 1....
## 3: 0.1362468 0.3150882 0.19381330 -0.3640706 -1.3431389    31 76199, 1....
## 4: 0.7779661 0.8806609 0.04959020  0.2516324  0.7382131    17 20893, 5....
## 5: 0.3884892 0.5913108 0.07511816  0.2469065  1.0473836   106 60406, 1....
## 6: 0.4101695 0.6090340 0.08085892  0.3607407  1.0583037    17 60406, 2....