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Hypergeometric test with no phenotype

In case if you only have a list of genes and want to do hypergeometric test with gene sets having known functions, HTSanalyzeR2 provides an interface to realize it. Since phenotype is only used as background genes in hypergeometric test, you can manually set all the genes of that species as phenotype and give them a pseudo value to fit HTSanalyzeR2 as below:

data(d7)
hits1 <- d7$id[1:200]
## set all the coding genes of Homo sapiens as phenotype
allgenes <- keys(TxDb.Hsapiens.UCSC.hg19.knownGene, keytype = "GENEID")
## change Entrez ID to gene name to keep consistent with hits
allgenes <- mapIds(org.Hs.eg.db, keys = allgenes,
                   keytype = "ENTREZID", column = "SYMBOL")
                   
## give phenotype a pseudo value to fit for HTSanalyzeR2
phenotype <- rep(1, length(allgenes))
names(phenotype) <- allgenes

Then, you can use the artificial phenotype as gene background and your hits to perform hypergeometric test.

gsca <- GSCA(listOfGeneSetCollections=ListGSC,
             geneList=phenotype, hits=hits1)
## the following analysis is the same as before

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Customized gene set

When you have your own gene sets with specific functions and they do not belong to any GO terms, KEGG or MSigDB. In that case, you can set your customized gene set collection and follow the format of GO, KEGG and MSigDB gene set collections. An important thing here you need pay attention to is the ID of genes in the gene set collection must be Entrez ID.

## Suppose your own gene sets is geneset1 and geneset2
allgenes <- keys(org.Hs.eg.db, "ENTREZID")
geneset1 <- allgenes[sample(length(allgenes), 100)]
geneset2 <- allgenes[sample(length(allgenes), 60)]

## Set your custom gene set collection and make the format to fit HTSanalyzeR2
CustomGS <- list("geneset1" = geneset1, "geneset2" = geneset2)
## then the gene set collections would be as below:
ListGSC <- list(CustomGS=CustomGS)
## other part is the same as before

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An interface to fgsea package

HTSanalyzeR2 also provides an interface to fgsea package which proposes a novel algorithm for fast preranked gene set enrichment analysis using cumulative statistic calculation. Details please see fgsea (Sergushichev (2016)). To perform GSEA by fgsea instead of HTSanlayzeR2, users need to specifiy the parameter GSEA.by in analyze (for single data set) or analyzeGscaTS (for time series analysis) with fgsea. Otherwise, it will use HTSanalyzeR2 by default.

gsca4 <- analyze(gsca1,
                 para=list(pValueCutoff=0.05, pAdjustMethod="BH",
                           nPermutations=100, minGeneSetSize=150,
                           exponent=1),
                 doGSOA = TRUE, doGSEA = TRUE,
                 GSEA.by = "fgsea")

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Extract shared genes between enriched pathways and input gene list

Once you’ve finished the GSEA or hypergeometric test, you may be interested in some pathways and wonder which genes are shared by that pathway and you input gene list.

## Suppose you are interested in "growth factor activity" in 'Molecular Function',
## of Gene Ontology, We can retrieve the GO ID of this pathway:
GO_MFrslt <- getResult(gsca3)$HyperGeo.results$GO_MF
GOID <- rownames(GO_MFrslt[GO_MFrslt$Gene.Set.Term ==
                                             "growth factor activity", ])
                                             
## Then get the genes in this pathways
pathway_gene <- GO_MF[[GOID]]

## change Entrez ID to gene symbol
pathway_gene <- mapIds(org.Hs.eg.db, keys = pathway_gene,
                       keytype = "ENTREZID", column = "SYMBOL")
## 'select()' returned 1:1 mapping between keys and columns

## get the shared genes between this pathway and your input gene list
intersect(pathway_gene, hits)
## [1] "CTGF"    "EFEMP1" "FGF7"  "IGF1"  "INHBA" "NOV"  "OGN"
## [8] "CLEC11A" "CXCL12" "TGFB3" "THBS4" "VEGFC" "DKK1" "PDGFC"

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