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Gene Set Enrichment Analysis and plots

2 messages · Anas Jamshed, Bert Gunter

Anas Jamshed
# 
I have done this analysis:

#You can select Working Directory according to your choice
setwd("D:")

#Check Working Directory
getwd()

#Creation of object(folder) exdir
exdir <- path.expand("~/GSE162562_RAW")
dir.create(exdir, showWarnings = FALSE)

URL <- "
https://ftp.ncbi.nlm.nih.gov/geo/series/GSE162nnn/GSE162562/suppl/GSE162562_RAW.tar
"
FILE <- file.path(tempdir(), basename(URL))

#Downlaod the Raw Data from GEO
utils::download.file(URL, FILE, mode = "wb")

#unzip the files and storing them in GSE162562_RAW folder which we created
already
utils::untar(FILE, exdir = exdir)

#Check your GSE162562_RAW folder after this , it must contains 108 samples
in compressed format

unlink(FILE, recursive = TRUE, force = TRUE)

#listing of samples
listed_files <- list.files(exdir, pattern=".gz", full.names=TRUE)


#/ load files into R:
loaded <- lapply(listed_files, function(x) read.delim(x, header=FALSE,
row.names = "V1"))

#/ bind everything into a single count matrix and assign colnames:
raw_counts <- do.call(cbind, loaded)
colnames(raw_counts) <- gsub(".txt.*", "", basename(listed_files))



#/ there is some nonsense in these files, probably due to the tool that was
used (HTSeq?),
#/ such as rows with names "__no_feature", lets remove that:
raw_counts <- raw_counts[!grepl("^__", rownames(raw_counts)),]

# / View raw_counts after filtering
raw_counts
#There are total 26364 genes after filtering

#Store Raw counts in CSV File
#I am sacing them in my Desktop.You can save according to your choice
write.csv(raw_counts,"C:\\Users\\USER\\Desktop\\countsdata.csv")

#load library
library(DESeq2)

#/ make a DESeq2 object. We can parse the group membership (Mild etc) from
the colnames,
#/ which are based on the original filenames:

dds <- DESeqDataSetFromMatrix(
  countData=raw_counts,

colData=data.frame(group=factor(unlist(lapply(strsplit(colnames(raw_counts),
split="_"), function(x) x[4])))),
  design=~group)


#View Deseq2 object
dds

#/ some Quality Control via PCA using the in-built PCA function from DESeq2:
vsd <- vst(dds, blind=TRUE)
#/ plot the PCA:
plotPCA(vsd, "group")


#/ extract the data:
pcadata <- plotPCA(vsd, "group", returnData=TRUE)

#view pca data
pcadata

#/ there is a very obvious batch effect, that we can correct, simply
everything that is greater or less than zero in the first principal
component, very obvious just by looking at the plot:
dds$batch <- factor(ifelse(pcadata$PC1 > 0, "batch1", "batch2"))

#/ lets use limma::removeBatchEffect to see whether it can be removed:
library(limma)
vsd2 <- vsd
assay(vsd2) <- removeBatchEffect(assay(vsd), batch=dds$batch)


#/ plot PCA again, looks much better. this means we modify our design to
include batch and group,
plotPCA(vsd2, "group")

#include batch and group both in design
design(dds) <- ~batch + group

#Running the differential expression pipeline
dds <- DESeq(dds)

#Building the results table
res <- results(dds)
res

#Saving Results in CSV file

write.csv(res , "dds.csv")

mcols(res, use.names = TRUE)

#We can also summarize the results with the following line of code, which
reports some additional information:
summary(res)

#Total 1236 DEGs have been found when we se p_value less than 0.1

table(res$padj < 0.01)

res.05 <- results(dds, alpha = 0.05)

summary(res.05)

table(res.05$padj < 0.05)

sum(res$pvalue < 0.05, na.rm=TRUE)

sum(!is.na(res$pvalue<0.05))

sum(res$padj < 0.1, na.rm=TRUE)

sum(res$padj < 0.05, na.rm=TRUE)

resSig <- subset(res, padj < 0.1)

head(resSig[ order(resSig$log2FoldChange),])

head(resSig[ order(resSig$log2FoldChange, decreasing = TRUE), ])

#Save DEGS at padj < 0.1
write.csv(resSig,"DEGs at 0.1.csv")

resSig0.05 <- subset(res, padj < 0.05)

#Save DEGS at padj < 0.01
write.csv(resSig0.05,"DEGs at 0.05.csv")


Now I want to do gene set enrichment analysis by using topgo library and
also want to make a heatmap and Venn diagram. Please help me
Bert Gunter
# 
Please read the posting guide linked below for how to post questions
that are likely to receive useful responses. "Help me do a gene
enrichment analysis" does not conform to the pg. We usually expect
posters to offer their best attempts, preferably in a reproducible
example ("a reprex" -- see here for discussion:
https://stackoverflow.com/questions/5963269/how-to-make-a-great-r-reproducible-example
), to provide help.

Also note that per the pg:
"For questions about functions in standard packages distributed with R
(see the FAQ Add-on packages in R), ask questions on R-help.
If the question relates to a contributed package , e.g., one
downloaded from CRAN, try contacting the package maintainer first. You
can also use find("functionname") and
packageDescription("packagename") to find this information. Only send
such questions to R-help or R-devel if you get no reply or need
further assistance. This applies to both requests for help and to bug
reports."

Finally, note that "topgo" is a Bioconductor **package** (not
"library"), and you should almost certainly post questions about its
use on their support site not here:
https://www.bioconductor.org/help/

(Do read their pg before posting, of course)

Bert Gunter

"The trouble with having an open mind is that people keep coming along
and sticking things into it."
-- Opus (aka Berkeley Breathed in his "Bloom County" comic strip )
On Mon, Aug 30, 2021 at 9:22 AM Anas Jamshed <anasjamshed1994 at gmail.com> wrote: