Running pathfindR package generates an error message

[obearap]

I'm using the pathfindR package to perform PSEA on a set of DE proteins from a proteomics experiment. If I understand the package correctly one of the outputs should be a dataframe which I have called PSEA_results.

When I run the package that dataframe doesn't get created and I get the following error message.

Error in (function (filename = "Rplot%03d.png", width = 480, height = 480, : invalid 'filename'

This is a new one on me. My code is below.

library ("KEGGREST")
library ("KEGGgraph")
library ("org.Hs.eg.db")
library ("pathfindR") 
library ("tidyverse")
library ("tidyr")
library ("dplyr")
library ("magrittr")

#remove all previous file
rm(list=ls())

#data summary needs pvalues added

#Data Summary of Progenesis Data, Enriched GO Terms and Enriched KEGG Pathways
attached_CHO_all_data <- read.csv("C:/PETER PROJECT/3d. Mass Spec Processing/Results of R Analysis/Data Summary/Effect of Culture Format/WCL Analysis/DE Cutoff equal 1.2/0% Attached v 0% Suspension/DE 1-2 0% Attached v Susp WCL Apr19 (CHO Database) - All Data.csv")

A_v_S_Comp_0rd <- read.csv("C:/PETER PROJECT/3d. Mass Spec Processing/Results of R Analysis/DE Normalised Abundance Data/Effect of Culture Format/WCL Analysis/DE Cutoff equal 1.2/0% Attached v 0% Suspension/DE 1-2 0% Attached v Susp Normalised Abundance Data WCL Apr19.csv")
colnames(A_v_S_Comp_0rd)

#find the average of each row relating to the 4 replicates of attached cells
Aver_A0 <- rowMeans(data.frame(A_v_S_Comp_0rd$A0b, A_v_S_Comp_0rd$A0c, A_v_S_Comp_0rd$A0d))
Aver_A0 <- data.frame(A_v_S_Comp_0rd$description, Aver_A0)

#find the average of each row relating to the 4 replicates of suspension cells
Aver_S0 <- rowMeans(data.frame(A_v_S_Comp_0rd$S0a, A_v_S_Comp_0rd$S0b, A_v_S_Comp_0rd$S0c, A_v_S_Comp_0rd$S0d))
Aver_S0 <- data.frame(A_v_S_Comp_0rd$description, Aver_S0)

#Divide each row of dataframe Aver_S5 by the corresponding row in dataframe Aver_A5 
FC <- cbind(Aver_S0[1],round(Aver_S0[-1]/Aver_A0[-1],3))
names(FC)[2] = "FC"

#Get Log2 0f A0/S0            
Log2FC = log2(FC$FC)

#select desired columns
PSEA_1<-  data.frame(A_v_S_Comp_0rd$description, Log2FC, A_v_S_Comp_0rd$Anova..p)

#spilt column 1 in the dataframe PSEA_rd into 2 separate columns using the phrase refseq: as the spilt and call it PSEA_2
PSEA_2<- tidyr::separate (PSEA_1, 1, into=c("HGNC_Symbol", "Refseq"), sep = "Refseq:")

#further spilt column 6 in the dataframe PSEA_2 into 2 separate columns using = as the spilt and call it v7
PSEA_3<- tidyr::separate (PSEA_2, 1, into=c("gene", "HGNC_Symbol"), sep = "=") 

#remove unwanted columns
PSEA_4 = subset(PSEA_3, select = -c(gene,Refseq))
PSEA_rd <- distinct(PSEA_4,HGNC_Symbol, .keep_all= TRUE)

#trim white space from protein names and make a dataframe
PSEA_rd$HGNC_Symbol <- trimws(PSEA_rd$HGNC_Symbol, which = "right", whitespace = "[ \t\r\n]")

#rename columns and sort on Gene.Symbol
names(PSEA_rd)[1] <- "Gene.Symbol"
names(PSEA_rd)[2] <- "LogFC"
names(PSEA_rd)[3] <- "adj.P.Val"
PSEA_rd <- arrange(PSEA_rd, Gene.Symbol, by_group = FALSE)
head(PSEA_rd)
str(PSEA_rd)

# PSEA_rd is the raw data file for PSEA
data(PSEA_rd)

#set a directory to store the results in. The default directory is is pathfindeR_Results
getwd()

PSEA_results <- run_pathfindR(PSEA_rd, output_dir = ("C:/PETER PROJECT/3d. Mass Spec Processing/Results of R Analysis/pathfindR/Culture Format/WCL/0% Attach v 0% Susp")) 

    #write.csv (PSEA_results, file = "C:/PETER PROJECT/3d. Mass Spec Processing/Results of R Analysis/pathfindR/Effect of Culture Format/WCL Analysis/0% Attached v 0% Suspension/DE Cutoff equal 1.2/PSEA_Attached_0.csv")

The data frame PSEA_rd has over 1000 lines in it so I don't propose to reproduce it here. However, just so people can see the types of data it contains the output from str(PSEA_rd) is below.

'data.frame':   1171 obs. of  3 variables:
 $ Gene.Symbol: chr  "AAAS" "AACS" "AARS" "AATF" ...
 $ LogFC      : num  -0.648 0.82 1.837 -0.492 -1.114 ...
 $ adj.P.Val  : num  7.15e-05 4.59e-03 4.71e-05 1.38e-02 1.00e-02 ...

I did try one other thing. I ran

PSEA_results <- run_pathfindR(PSEA_rd)

The result was the same error message which hopefully rules out that the issue is caused by the directory structure I want to store the data in.

Thanks in advance for any suggestions..

[egeulgen]

Do you mind sharing the R console output, including the error line?
This is curious because pathfindR does not contain the line that you shared as the error code.
If visualization is not immediately necessary and your aim is to have enrichment results, I would try:

PSEA_results <-  run_pathfindR(input = PSEA_rd, visualize_enriched_terms = FALSE, plot_enrichment_chart = FALSE)

visualize_enriched_terms = TRUE saves (by default) annotated KEGG diagrams and plot_enrichment_chart = TRUE produces a graphical summary of enrichment results. Both can be created separately.

Let me know if running the command above works. If not, please share the console output.

[obearap]

That line of code worked perfectly. Thanks. As I am new to this whole field (Im a final year PhD student who ended up in BioInformatics) could you provide the code to produce the annotated KEGG diagrams and the enrichment results?

Also is it possible to produce annotated KEGG diagrams for all the pathways identified? Sometimes the pathways of interest aren't the ones which are most significantly enriched.

[egeulgen]

hey @obearap,

you may generate visualizations via the function visualize_terms():

processed_input_df <- input_processing(input_df)
visualize_terms(result_df = output_df, input_processed = processed_input_df)

This produces (by default) annotated KEGG diagrams for all the pathways in output_df. input_processed would be the processed input_df (that you used with run_pathfindR())