Dear All,

I'm experiencing a problem while using your super-useful packages DOSE and ClusterProfiler.
I try to create a for loop to process multiple genelists in batch. The script works generating the result-tables but fails to find the enrichment objects (namely ego's,ggo's, kk and kk2 that you'll see in the following script). It seems to me that they are unavailable after the table creation and can’t be found by the graphical functions (barplot, dotplot, enrichmap or cnetplot). If I run it out of the for loop, it works smoothly with a single list, I mean the exact same commands.

Here is the error I get when graphical functions (dotplot in this case) fail:

Error in dotplot(egoBP) :

error in evaluating the argument 'object' in selecting a method for function 'dotplot':

Error: object 'egoBP' not found

Here is my script in case you can tell me what can be the problem:


###############ClusterProfiler#############################

#install.packages(c("DOSE", "clusterProfiler"),repos="http://www.bioconductor.org/packages/devel/bioc")
# source("https://bioconductor.org/biocLite.R")
# biocLite (c("tidyr","pathview","Rgraphviz","RDAVIDWebService"))
#install.packages("rJava")
library(DOSE)
library(clusterProfiler)
#library(RDAVIDWebService)

x<-setwd("C:/R_data/Folder/")

file.list <- as.list(list.files(".", pattern = "*.tsv$"))
file.list

for(i in 1:length(file.list))
{
  #create the new output directory list
  dir<- gsub("\\.tsv$", "", file.list[[i]])
  file.path<-x
  dir.create(file.path(dir))
 
  #read the table
  geneList2<- read.table(file.list[[i]]) #For loop
  head(geneList2)
  str(geneList2)

  #generate vector
  gene.vector=geneList2$V2
  names(gene.vector)=geneList2$V1
  head(gene.vector)
 
  vlist<-gene.vector
  head(vlist)
  str(vlist)

  listaGenes <- as.vector(geneList2[,1])#
  head(listaGenes)
  genes<-as.character(listaGenes)#list of genes

  #Gene Ontology Classification Examples
  ggoBP <- groupGO(gene     = genes,
                   organism = "mouse",#organism = "human"
                   ont      = "BP",
                   level    = 3,
                   readable = TRUE)
  head(summary(ggoBP))
  write.csv(summary(ggoBP), file=(paste(file.path, dir,"GOc_GOBp_table.csv", sep="/")))
 
  #GO over-representation test
  egoBP <- enrichGO(gene          = genes,
                    universe      = allEntrez,
                    organism = "mouse",#organism = "human"
                    ont           = "BP",
                    pAdjustMethod = "BH",
                    pvalueCutoff  = 0.1,#pvalueCutoff  = 0.01,
                    qvalueCutoff  = 0.05,
                    readable      = TRUE)
  head(summary(egoBP))
  write.csv(summary(egoBP), file=(paste(file.path, dir,"Overrep_GOBP_table.csv", sep="/")))
 
  #GO Gene Set Enrichment Analysis
  ego2BP <- gseGO(geneList     = vlist,
                  organism = "mouse",#organism = "human"
                  ont          = "BP",
                  nPerm        = 1000,
                  minGSSize    = 5,#120,
                  pvalueCutoff = 0.5,#pvalueCutoff = 0.01,
                  verbose      = FALSE)
  write.csv(summary(ego2BP), file=(paste(file.path, dir,"gseGOBP_table.csv", sep="/")))
 
  #KEGG over-representation test
  kk <- enrichKEGG(gene         = genes,
                   organism     = "mouse",#organism     = "human",
                   pvalueCutoff = 0.05,
                   readable     = TRUE)
  #use_internal_data = TRUE)
  head(summary(kk))
  write.csv(summary(kk), file=(paste(file.path, dir,"enrichKEGG_table.csv", sep="/")))
 
  #Graphical Visualization Examples
  #Barplot
  pdf(paste(file.path, dir, "Enrichment_barplot.pdf", sep="/"))
  barplot(egoBP, showCategory=8,font.size=5)
  dev.off()

  #Dotplot
   pdf(paste(file.path, dir, "Enrichment_dotplot.pdf", sep="/"))
  dotplot(egoBP)
  dev.off()
 
  #Enrichmap
   pdf(paste(file.path, dir, "Enrichment2_Map.pdf", sep="/"))

  enrichMap(egoBP,vertex.label.cex=1.2,layout=igraph::layout.kamada.kawai)
  dev.off()
 
  #cnetplot
  pdf(paste(file.path, dir, "Enrichment_Cnet.pdf", sep="/"))
  cnetplot(egoBP, categorySize="pvalue", foldChange=vlist)
  dev.off()
 
  #Networks
  pdf(paste(file.path, dir, "Enrichment_Networks.pdf", sep="/"))
  plotGOgraph(egoBP)
  dev.off()
}

 

> sessionInfo()

R version 3.2.2 (2015-08-14)

Platform: x86_64-w64-mingw32/x64 (64-bit)

Running under: Windows 7 x64 (build 7601) Service Pack 1

locale:

[1] LC_COLLATE=Spanish_Spain.1252  LC_CTYPE=Spanish_Spain.1252  

[3] LC_MONETARY=Spanish_Spain.1252 LC_NUMERIC=C                 

[5] LC_TIME=Spanish_Spain.1252   

attached base packages:

 [1] grid      parallel  stats4    stats     graphics  grDevices utils   

 [8] datasets  methods   base    

other attached packages:

 [1] pathview_1.10.1       org.Hs.eg.db_3.2.3    Rgraphviz_2.14.0    

 [4] graph_1.48.0          SparseM_1.7           GO.db_3.2.2         

 [7] org.Mm.eg.db_3.2.3    AnnotationDbi_1.32.0  IRanges_2.4.4       

[10] S4Vectors_0.8.3       Biobase_2.30.0        BiocGenerics_0.16.1 

[13] clusterProfiler_2.4.3 DOSE_2.8.3            RSQLite_1.0.0       

[16] DBI_0.3.1           

loaded via a namespace (and not attached):

 [1] Rcpp_0.12.2       plyr_1.8.3        XVector_0.10.0    tools_3.2.2     

 [5] zlibbioc_1.16.0   digest_0.6.8      lattice_0.20-33   gtable_0.1.2    

 [9] png_0.1-7         KEGGgraph_1.28.0  igraph_1.0.1      curl_0.9.4      

[13] proto_0.3-10      topGO_2.22.0      stringr_1.0.0     httr_1.0.0      

[17] Biostrings_2.38.2 qvalue_2.2.2      R6_2.1.1          XML_3.98-1.3    

[21] GOSemSim_1.28.2   ggplot2_1.0.1     reshape2_1.4.1    DO.db_2.9       

[25] magrittr_1.5      scales_0.3.0      MASS_7.3-45       splines_3.2.2   

[29] KEGGREST_1.10.1   colorspace_1.2-6  labeling_0.3      stringi_1.0-1   

[33] munsell_0.4.2

I can't reproduce your issue.

Although you should explicitly call print(dotplot()) since the function return ggplot object, it won't complain object not found if it is exists.

Error in dotplot(egoBP) :

error in evaluating the argument 'object' in selecting a method for function 'dotplot':

Error: object 'egoBP' not found

object not found! this is explictly.

for(i in 1:length(file.list))
{

print(i)

...

}

Maybe you can print i before processing the file to help identifying which file have the issue.

Thank you Guangchuang Yu!

Explicitly calling print(dotplot()) and print(barplot()), did the trick and allowed me to obtain the graphics inside the FOR loop.

Thank you also for this remarkable package

Best wishes

JL