Question

ssGSEA for beginners

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4.6 years ago

shweta.johari13 ▴ 30

Greetings! I have a one gene expression input file (.gct) and one geneset (.gmt) file and need to run ssGSEA for this set. How do I proceed with this? Please do help me.

Thanking you.

RNA-Seq R ssGSEA • 12k views

ADD COMMENT • link 4.6 years ago by shweta.johari13 ▴ 30

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What have you tried?

ADD REPLY • link 4.6 years ago by Ram 43k

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Respected Sir,

I downloaded this software from https://github.com/broadinstitute/ssGSEA2.0 as well as downloaded R studio.

In addition, I found the following:

R-GUI / RStudio The script ssgsea-gui.R requires little or no knowledge of R or on how to use the command line. Input files and databases can be specified via Windows file dialogs that will be automatically invoked. The first dialog lets you choose a folder containing input files in GCT v1.2 or GCT v1.3 format. The script loops over all GCT files in this directory and runs ssGSEA on each file separately. The second dialog window lets the user choose one or multiple gene set databases in GMT format such as MSigDB. A current version of MSigDB databases can be found in the db subfolder.

Windows OS To run the script source it into a running R-session.

RStudio: open the file and press 'Source' in the upper right part of the editor window R-GUI: drag and drop this file into an R-GUI window iOS/MAC In order to invoke file dialogs as decribed above, the XQuartz X Window System is required. Once installed ssgsea-gui.R can be sourced into an R session.

Command line For integration of ssGSEA2.0/PTM-SEA into your own analysis pipelines we recommend to use the ssgsea-cli.R script which has been successfully tested on Windows, Mac and Linux OS. Please see ssgsea-cli.R --help for instructions.

ADD REPLY • link 4.6 years ago by shweta.johari13 ▴ 30

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Hey, thank you for explaining, but where, exactly, is the difficulty? You seem to have already followed the instructions.

ADD REPLY • link 4.6 years ago by Kevin Blighe 87k

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Do you know R? What sort of experience do you have with the command line? Why do you wish to use ssGSEA? What is the biological question you're trying to answer?

ADD REPLY • link 4.6 years ago by Ram 43k

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Respected Sir,

I know what a command line is. I have Just joined a Ph.D. Program and my experiment requires to use ssGSEA. The question we are trying to know is the immunological markers. Different sections of the Tumor will be cut, the rna will be extracted and sequencing will be done and post that GSEA. In addition to that, ssGSEA is proposed to be done. Thereby, it is an urgent need to learn this.

ADD REPLY • link 4.6 years ago by shweta.johari13 ▴ 30

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That's great, I'd recommend using the command line as it is a lot more flexible than GUI stuff. Run ssgsea-cli.R --help (once you've installed R) and start playing with it from there.

ADD REPLY • link 4.6 years ago by Ram 43k

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Respected Sir.

I ran the following script and an error was generated

RScript ssGSEA2.0-master/ssgsea-cli.R
Error: unexpected symbol in "RScript ssGSEA2.0"

After installing R, I put the command line but this error was generated.

ssgsea-cli.R
Error: object 'ssgsea' not found
ssgsea-cli.R --help 
Error: object 'ssgsea' not found

ADD REPLY • link updated 4.6 years ago by Ram 43k • written 4.6 years ago by shweta.johari13 ▴ 30

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If not the CLI, then this sequence of commands should work for the GUI (?)

git clone https://github.com/broadinstitute/ssGSEA2.0
cd ssGSEA2.0/
R
source('ssgsea-gui.R')

ADD REPLY • link 4.6 years ago by Kevin Blighe 87k

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Respected Sir,

After puting the script in R, the following was shown:

git clone https://github.com/broadinstitute/ssGSEA2.0
Error: unexpected symbol in "git clone"
> cd ssGSEA2.0/
Error: unexpected symbol in "cd ssGSEA2.0"
>     R
Error: object 'R' not found
> source('ssgsea-gui.R')
Error in file(filename, "r", encoding = encoding) : 
  cannot open the connection
In addition: Warning message:
In file(filename, "r", encoding = encoding) :
  cannot open file 'ssgsea-gui.R': No such file or directory

ADD REPLY • link updated 4.6 years ago by Kevin Blighe 87k • written 4.6 years ago by shweta.johari13 ▴ 30

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Run these outside R, in a terminal:

git clone https://github.com/broadinstitute/ssGSEA2.0

cd ssGSEA2.0/

R

Then, R will open. Then run:

source('ssgsea-gui.R')

ADD REPLY • link 4.6 years ago by Kevin Blighe 87k

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Respected Sir,

Thank you so much for all the help.

I have done the following:

From https://github.com/broadinstitute/ssGSEA2.0 I have downloaded the zip file of ssGSEA.

Then extracted those files in a folder.

Simultaneously I opened R and set my working directory,

Then I installed the packages of pacman.

Post that I sourced in the .gct and .gmt file of the example data provided by the software.

The run was complete and I received the output as

rank-plots
signature.gct
parameters
combined.gct
fdrvalues.gct
p values
pvalues.gct
scores.gct

Thereby it is running.

However, unfortunately, when I am trying my mouse data .gct file, the following error is being shown which I think indicates that my .gct file is in the incorrect format.

Running ssSGEA on: FFPEAGCTfinal.gct 

parsing as GCT v1.2
C:\Users\pc\Desktop\17 oct 2019 try1\running my data/FFPEAGCTfinal.gct 15859 rows, 1 cols, 0 row descriptors, 0 col descriptors
parsing as GCT v1.2
C:\Users\pc\Desktop\17 oct 2019 try1\running my data/FFPEAGCTfinal_unique.gct 15859 rows, 1 cols, 0 row descriptors, 0 col descriptors
Error in ssGSEA2(input.ds, gene.set.databases = gene.set.databases, sample.norm.type = sample.norm.type,  : 
  No overlap to any gene sets found in your data set!
Possible reasons: 1) organism other than human; 2) wrong format of gene names/site ids!
In addition: Warning messages:
1: In dir.create(date.str) : '2019-10-17' already exists
2: In ssGSEA2(input.ds, gene.set.databases = gene.set.databases, sample.norm.type = sample.norm.type,  :

 Error in ssGSEA2(input.ds, gene.set.databases = gene.set.databases, sample.norm.type = sample.norm.type,  : 
  No overlap to any gene sets found in your data set!
Possible reasons: 1) organism other than human; 2) the wrong format of gene names/site ids!

ADD REPLY • link updated 4.6 years ago by Ram 43k • written 4.6 years ago by shweta.johari13 ▴ 30

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Yes. The data is for mouse and not human. In addition, the .gct file is singular column that contains 15859 rows.

ADD REPLY • link 4.6 years ago by shweta.johari13 ▴ 30

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It is likely that your gene IDs are not matching. Mouse (Mus musculus) gene names are different from Human (Homo sapiens). You will need to convert them.

ADD REPLY • link 4.6 years ago by Kevin Blighe 87k

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Respected Sir,

Yes. I made a silly mistake in regards to the .gmt file and while correcting that I have received another error in regards to the .gct file. As it is a single-sample gene set enrichment analysis, I am trying to understand why one column that represents a specific experiment (e.g. treatment of cell line MCF7 with a small-molecule drug) and each row represents features (e.g. genes) that are measured in the assay is not being read.

ADD REPLY • link 4.6 years ago by shweta.johari13 ▴ 30

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Running ssSGEA on: FFPEAGCTfinal.gct 

parsing as GCT v1.3
C:\Users\pc\Desktop\shweta\attempt1/FFPEAGCTfinal.gct 15859 rows, 1 cols, NA row descriptors, NA col descriptors
Error in ssGSEA2(input.ds, gene.set.databases = gene.set.databases, sample.norm.type = sample.norm.type,  : 


Error importing GCT file using 'cmapR::parse.gctx()'. The GCT file doesn't seem to be in the correct format!
Please see take a look at https://clue.io/connectopedia/gct_format for details about GCT format.

Error message thrown by 'cmapR::parse.gctx()':

In addition: Warning message:
In FUN(X[[i]], ...) :

 Error in ssGSEA2(input.ds, gene.set.databases = gene.set.databases, sample.norm.type = sample.norm.type,  : 


Error importing GCT file using 'cmapR::parse.gctx()'. The GCT file doesn't seem to be in the correct format!
Please see take a look at https://clue.io/connectopedia/gct_format for details about GCT format.

Error message thrown by 'cmapR::parse.gctx()':

ADD REPLY • link updated 4.6 years ago by Ram 43k • written 4.6 years ago by shweta.johari13 ▴ 30

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Hi,

The error messages are quite clear and provide a good lead to follow. Please try solving these on your own - that's how all of us learn. Asking for help with each step is asking for hand-holding/spoon-feeding. It is easy right now but will only harm learning in the long term.

As a side note, please use the formatting bar (especially the code option) to present your post better. You can use backticks for inline code (`text` becomes text), or select a chunk of text and use the highlighted button to format it as a code block. I've done it for you this time.
code_formatting

ADD REPLY • link 4.6 years ago by Ram 43k

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Respected Sir,

Yes, sir, I understand and I did hope this platform was for asking the doubts (I did not know there was a limit) and clearing it. However, I completely understand your point of view on the spoon-feeding aspect. I sincerely apologize for my constant replies asking for help. Thank you for all the help. I shall surely learn. Yes sir, in the future, I shall use the text format the way it should be. Thanking you again.

ADD REPLY • link 4.6 years ago by shweta.johari13 ▴ 30

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Crossposted at SO:

ssGSEA for those who do not know R

ADD REPLY • link 4.6 years ago by zx8754 11k