Just leaving a comment hopefully for people that are trying to use it recently. The Expression Dataset File by default is no longer like that: just remove the first 2 rows (starting with the row: Name "tab" Description "tab" ...) I did that and everything run smoothly! You can also see it as the last example in the user guide web page (did they change the default standard?)
Hi Magaraju-India, heatmaps are included in the outputs, but they don't show all the genes. This is a better tool if you just want heatmaps: www.heatmapper.ca/
@@GenomicsGurus Madam, I would like to have an output represented at 32min:49 sec to 33min.30 sec of your video. Please let me know the process with options. Thank you.
Hi, you don't have to select any options -the heat maps appear automatically underneath the first table. However, sometimes they don't display as it depends on the html file being able to access the image file eg I had trouble when I saved the output to an online location (one drive) and this was solved when I saved the outputs to my c drive. If they still don't load up, the heat maps are saved as individual pictures in the folder where you save the output, so go to your file manager, find the folder, and view them from there.
Thank you for this helpful tutorial! I like how you explained all the output metrics in detail. I had zero encounters with RNA seq data analysis and within a few hours, I managed to compare my gene sets of interest in my experimental groups.
I have a note about what you said at 3:33 The genes are ranked based on their P. Value and fold change, so saying based on counts isn't entirely true. Thank you so much for the video it's really helpful.
There is a mistake in the explanation. Do not add the #1.2 plus number of genes and columns in the file when saving as TXT, it only works when using GCT.
Thank you Dr. West! Good job, great tutorial. I even liked your warm voice and your accent... it sounds like you are American, but maybe it is the Scottish accent... I've never been in Scotland, I couldn't say.
I'm glad you found the tutorial useful! I think my accent is mainly Scottish, but there's probably a twinge of the eastern USA and north west England as well ;)
3:20 my understanding from reading the paper is that the genes ranking is based on some measure of correlation between the gene expression and the phenotype class, not just the average of the expressions for that gene. I don't think they are the same.
Thank you, was helpful to understand and perform GSEA. I would like you to cover network construction between miRNA-mRNA expression profiles using Cytoscape
Hi Katherine...........excellent explanation of GSEA for beginners.......... Can you please cover using ClueGo plugin in Cytoscape for building PPI maps..........
@@GenomicsGurus Thank you, it would be great to see the integrative analysis of RNA-Seq and Chip-Seq. EaSeq would be a good option for such a analysis but unfortunately I am not an expert in bioinformatics field.
This video is really helpful. I learned a lot from it. I was wondering do you have an example for time series analysis. Since the GSEA website doesn't talk too much about it, I have no idea to start the time series analysis.
@@GenomicsGurus Dear Madam, Can we change color of the heat maps. It is all blue and red whilst I run GSEA and check the output folder. Is it possible. Please let me know madam. Thank you.
@@scienceforus8669 There is an option in the advanced settings to save the heatmap data as a gct file. You could then import that into a web tool for heatmaps where you can customise the colours.
I'm doing my RNA Seq data analysis, and I've got the differentially expressed genes in an excel sheet. I would like to do pathway analysis to see which pathways are differentially regulated now. I don't know how to do that, I hope this tutorial helps me! Thanks!
I need to add one important point here is that the file format of the expression data mentioned here should have a .gct extension. It shouldn't be a .txt extension. tab-delimited file doesn't take the the two rows at the top
Glad you found it useful. I'm not sure when I'll get the time to do one on pre-ranked GSEA. Is that something you want to try? There's only a couple of things that are different, I think - I can write them down for you.
@@sumitpaliwal1540 This is the link describing the rnk format your file needs to be in: software.broadinstitute.org/cancer/software/gsea/wiki/index.php/Data_formats#RNK:_Ranked_list_file_format_.28.2A.rnk.29 This method doesn't tolerate duplicate gene names in your list, though, which is a problem when I use Ensemble IDs, as each gene may have several different Ensemble gene IDs. If you are using Ensemble IDs I suggest you convert to gene symbols first, then sort by name (in excel) to identify any duplicates which you can then remove. If you're still having trouble, email me: katherine.west at glasgow.ac.uk and I'll have a look at your file
@@GenomicsGurus I do not have any duplicates in the list. The error I get is "After pruning, none of the gene sets passed size threshold". I can send you the screenshot and the file.
Hello, It is really a great video. I have a question related to validation of repurposed drugs using GSEA. Actually, I am a beginner and want to do this. Can you please guide me how can I validate the drugs that I have using GSEA? Looking forward for your response
Firstly, thank you for the tutorial, I have found it really helpful. I have a question regarding to what can be concluded from the enrichment. I have performed a preranked method and the results showed an enrichment in the pathway, however one of the genes that is highly expressed has an inhibitory function in the pathway. So my question is if the ES shows only the enrichment of the genes (either activators or inhibitors) or also the directionality of the pathway. Thank you in advance.
Think it only tells you about the pathway in general and not on the individual genes in the pathway. In case you want to know about the directionality for individual genes, probably you would have to check your individual genes and the fold change for that gene from your DE table.
Amazing tutorial. My question is, if i have RNA-seq results giving different expressed genes for different cell lines and I want to compare in order to check for example which cell line expresses genes (significant upregulated enrichment) associated with angiogenesis. In this case, in my expression dataset file, the first column would include ENS id's for these genes, but I have sometimes completely different genes in different cell lines (with 3 samples per cell line), so what is the appropriate way to organize this data?
Great video! Thanks a lot for the explanation. I was wondering if you know how to use a continuous phenotype label for a time course actually. I tried following the user guide for that but I couldn't really do it. Thanks :)
I've not tried it myself, but it doesn't look too complicated. There are lots of ways to make mistakes with these input files, though, as my students will testify! If you want to email your files to me, I can have a look at them.....katherine.west at glasgow.ac.uk
This content is a treasure. Thank you so much Dr. West. Just in case someone reads my comment: I have doubts about which values I need to put in the Expression data set file (10:37). I just have two groups and I have the "Raw comparison" values and other corrected values such as reads per kilobase per million (RPKM) and Transcripts Per Kilobase Million (TPM). Which one should I use? Another question: My data comes from a RNA-seq analysis of Mus musculus cells. Which Chip platform (23:40) should I choose? Many thanks! : )
Hi Joan, sorry for the slow reply. TPM is probably the best dataset to use. Your genes are probably named as ensemble gene IDs (ENSMUSGXxxx) so "mouse ensembl gene ID human orthologs" with the latest number would be the right chip platform to use.
Hi Dr. West, great tutorial. I have one question looking for your help. When creating Phenotype labels file in excel, you mentioned to save as tab-delimited text file with the extension.cls. How to do this? Excel does not have an option to save a file as cls file. Thanks.
Thank you very much for your tutorial. I have one question: Have you previously filtered the expression data? I mean, for example, applying a given Fold Change and filtering it by P-value.
Excellent tutorial and this made my concepts so clear. Just wondering if I can use GSEA for RNA seq analysis of any other organism. I am struggling with some Mycobacterium tuberculosis RNA seq data and could not find the GMT files for it. Is there any database from where I can download the GMT files for Mycobacterium tuberculosis.
I'm not sure if there are M. tuberculosis databases out there, but it's easy enough to make your own GMT files - it's just a list of gene IDs with a couple of rows at the top. You can download any current GMT file to see the format. I suggest you use the literature to find the genes associated with the pathway/phenotype you are interested in and make your own list. Good luck!
Hi, thanks for the tutorial. I had some trouble downloading the GSEA software so I ended up using the GenePattern UI. Is there any downside to using this website instead of the GSEA desktop application? Thanks
You shouldn't need the gene ID - choose a gene symbol chip platform instead of a gene ID chip platform when running GSEA. The long answer to your question is that you can download a file from ensembl that lists gene ID and gene symbols, and you can use vlookup in excel to look up IDs for known gene symbols and vice versa.
Just leaving a comment hopefully for people that are trying to use it recently.
The Expression Dataset File by default is no longer like that: just remove the first 2 rows (starting with the row: Name "tab" Description "tab" ...)
I did that and everything run smoothly! You can also see it as the last example in the user guide web page
(did they change the default standard?)
It would be great if example files were available to learn from, including the changes you indicated.
such a enjoyment to listening this clear explanation with such clear English speaking
I literally didnt like anlaysing the RNAseq data for my project samples for the past 1 year. After seeing your video, it was eye-opening.
Glad you found it helpful!
@@GenomicsGurus . Oh yes! definitely :)
It was about time that I was searching for a "real" GSEA tutorial. Thanks very much!
Hope you get some useful information from it!
This was really a really well done, in depth walkthrough of GSEA!
Thanks Anthony - glad you liked it
An excellent presentation and made GSEA understand quickly. I recommended this to my colleagues and co-researchers- Very well done.
Glad you found it helpful!
@@GenomicsGurus Madam, please le me know how to generate heat maps with this software. Please let me know what options should be used. Thank you.
Hi Magaraju-India, heatmaps are included in the outputs, but they don't show all the genes. This is a better tool if you just want heatmaps: www.heatmapper.ca/
@@GenomicsGurus Madam, I would like to have an output represented at 32min:49 sec to 33min.30 sec of your video. Please let me know the process with options. Thank you.
Hi, you don't have to select any options -the heat maps appear automatically underneath the first table. However, sometimes they don't display as it depends on the html file being able to access the image file eg I had trouble when I saved the output to an online location (one drive) and this was solved when I saved the outputs to my c drive. If they still don't load up, the heat maps are saved as individual pictures in the folder where you save the output, so go to your file manager, find the folder, and view them from there.
Thank you for this helpful tutorial! I like how you explained all the output metrics in detail. I had zero encounters with RNA seq data analysis and within a few hours, I managed to compare my gene sets of interest in my experimental groups.
Great! Hope you found something interesting!
I have a note about what you said at 3:33
The genes are ranked based on their P. Value and fold change, so saying based on counts isn't entirely true.
Thank you so much for the video it's really helpful.
Thank you so much for the detailed tutorial. Its alot easier to understand than the user guide which misses out details on the input and ranking.
Glad it's helpful. Hope you get some useful results!
Very helpful and clear tutorial, really appreciate it
Finally a real tutorial! Thank you!
Mandatory viewing for using the tool, thank you!
This video is amazing, so far my favorite. Really clear and straightforward, I truly appreciate it! Great job! many thanks :)
Thanks!
I can't thank you enough! You made my day! 🙏🏻🙏🏻🙏🏻
Thank you! This was a great introduction to GSEA. I found it extremely helpful. I wish they did more tutorial like this for other software!
Glad it was helpful!
Such a wonderful and informative Illustration of GSEA. Thank you so much.
Glad you found it useful!
The tutorial was amazing and easy to follow. Well done and looking forward to future videos.
Great presentation
Thanks so much again for helping me with fixing my files. That was a huge support.
Thanks for such a great tutorial! I've been struggling a little to analyze my RNAseq data, but I hope with this info I'll be able to do it.
I hope you get some interesting results!
what a wonderful course! I do watched several before this one, this is the best!!!
Super helpful and clear tutorial! I appreciate you saved me a lot time to figure out how to do such analysis!
It's a very very very good tutorial to introduce GSEA!!
Thanks :) glad to have helped!
Awesome! This was extremely well done. I am a novice at NGS analysis and found this very understandable and helpful.
Glad you found it helpful. Good luck!
Thank you. That was concise and wonderfully analyzed .
Meanwhile your British accent is super!
Thanks for your kind feedback. Glad you found it useful!
Great video. Clear and easy to follow tutorial. Great job Doctor!
Glad it was helpful!
So much help. Thank you.
Glad it helped!
Great tutorial, many thanks, Dr. Katherine West.
Glad you found it useful :)
Thank you, this was a great tutorial! I was struggling with multiple errors before, but now everything runs smoothly. Good job :)
Great! Glad it helped you sort things out :)
Thank your so much for making the tutorial. It is really helpful.
Great!
Absolutely invaluable tutorial! Thank you for creating this!
There is a mistake in the explanation. Do not add the #1.2 plus number of genes and columns in the file when saving as TXT, it only works when using GCT.
Oh, that's good to know - thanks very much much!
Very important comment. When I removed genes and columns it works with TXT file.
I have a wish 🙏 god, please let her return to youtube to make her awesome videos 🙏 Amen 🙏 Love from Turkey My Teacher 🙏
Can't thank you enough for such an invaluable video!
Great Introduction to GSEA, Thank you very much
Nice and detailed presentation, fully understandable. I really loved this GSEA tutorial/introduction. +1 subscriber
Thanks for your kind feedback. Glad you found it useful
Excellent tutorial! Thank you very much! Would love to see more like this!
Thank you Dr. West! Good job, great tutorial. I even liked your warm voice and your accent... it sounds like you are American, but maybe it is the Scottish accent... I've never been in Scotland, I couldn't say.
I'm glad you found the tutorial useful! I think my accent is mainly Scottish, but there's probably a twinge of the eastern USA and north west England as well ;)
Excellent talk Katherine
Extremely helpful. Thank you very much.
Thank you for sharing your wisdom with us.
You're welcome. Do you have any questions about it?
no tanks, this is great.
Thanks for this tutorial….
Could you please make a video on Cytoscap??
Glad it was helpful. Cytoscape is on our list
Thank you so much, its enriched and fruitful video, thanks genius :)
3:20 my understanding from reading the paper is that the genes ranking is based on some measure of correlation between the gene expression and the phenotype class, not just the average of the expressions for that gene. I don't think they are the same.
Woww great all concepts are cleared now
Glad it was helpful.
Thank you, was helpful to understand and perform GSEA. I would like you to cover network construction between miRNA-mRNA expression profiles using Cytoscape
Glad you found it useful. We hope to cover Cytoscape when we get some time!
this is gold. thank you very much
Really great tutorial... Alot of info worth the time 😍
Glad you found it useful!
Very nicely done. Thank you for making this video.
We are pleased you found it useful Mo
This was a very helpful tutorial, Thank you.
Glad to hear that!
Thanks for sharing. Very helpful!
Glad it was helpful!
Hi Katherine...........excellent explanation of GSEA for beginners..........
Can you please cover using ClueGo plugin in Cytoscape for building PPI maps..........
Thanks Harish. We hope to cover Cytoscape in future
this is Brilliant! a thousand thank you!
Glad it was helpful!
Indeed very useful and well explained. Thank you!
Glad you found it helpful!
Very great explanation, thank god you made this video!!
Glad you found it useful!
Very nice tutorial. Thanks!
Glad it was helpful!
Thanks, this tutorial's really helpful for my work!
Amazing tutorial! Congrats!
Glad it was helpful!
Excellent, thank you!
Clear and highly helpful tutorial.
Amazing tutorial! Thank you very much!
Glad you found it helpful!
Thank you so much for the detailed tutorial!! Love it!
Glad to help!
This really helped me. Thanks.
A great tutorial! Thank you so much it is really helpful :)
Thanks! Hope your data is interesting :)
Nice Presentation 👍 Crystal Clear explanations Thanks a lot 😊, Would be Great to learn about time course analysis also!
Thanks for your feedback. We're pleased that you found it useful. There are more videos on this topic to follow so subscribe and look out for them 😀
An excellent tutorial and easy to follow. Thank you so much. Can you please give a tutorial on EaSeq open source software too ?
Thanks Pedram. Glad you found it useful. We will be covering ChIP-seq soon
@@GenomicsGurus Thank you, it would be great to see the integrative analysis of RNA-Seq and Chip-Seq. EaSeq would be a good option for such a analysis but unfortunately I am not an expert in bioinformatics field.
Useful presentation. Thanks
Glad it was helpful!
This video is really helpful. I learned a lot from it. I was wondering do you have an example for time series analysis. Since the GSEA website doesn't talk too much about it, I have no idea to start the time series analysis.
Amazing video, thanks!
Thank you@
Amazing tutorial! thank you!
Thanks Ariel. Glad you found it useful.
really help me out! many thanks!
Glad you found it useful!
Dear Madam, Please make some classes on "using R and GSEA". This video is great and I thoroughly enjoyed it. Thank you madam.
I'm not an R expert, so it will be a while before i can do that :) I hope you find some useful results from your research.
@@GenomicsGurus Dear Madam, Can we change color of the heat maps. It is all blue and red whilst I run GSEA and check the output folder. Is it possible. Please let me know madam. Thank you.
@@scienceforus8669 There is an option in the advanced settings to save the heatmap data as a gct file. You could then import that into a web tool for heatmaps where you can customise the colours.
@@GenomicsGurus Thank you Madam
I'm doing my RNA Seq data analysis, and I've got the differentially expressed genes in an excel sheet. I would like to do pathway analysis to see which pathways are differentially regulated now. I don't know how to do that, I hope this tutorial helps me! Thanks!
Great! Let us know how you get on!
Thank you, this is very clear
Glad it was helpful Jeannie!
I need to add one important point here is that the file format of the expression data mentioned here should have a .gct extension. It shouldn't be a .txt extension.
tab-delimited file doesn't take the the two rows at the top
Thanks for the feedback. This issue has come up already and is mentioned in the video description
Great class!!!
Glad you found it useful!
Good talk.. appreciate your efforts to help
Thanks for your kind feedback Raj!
Thanks a lot. Wonderful presentation. Can you do one on Preranked GSEA?
Glad you found it useful. I'm not sure when I'll get the time to do one on pre-ranked GSEA. Is that something you want to try? There's only a couple of things that are different, I think - I can write them down for you.
@@GenomicsGurus Yes. I have tried it a few times without success. The major issue is preparing a Preranked list .
@@sumitpaliwal1540 This is the link describing the rnk format your file needs to be in: software.broadinstitute.org/cancer/software/gsea/wiki/index.php/Data_formats#RNK:_Ranked_list_file_format_.28.2A.rnk.29
This method doesn't tolerate duplicate gene names in your list, though, which is a problem when I use Ensemble IDs, as each gene may have several different Ensemble gene IDs. If you are using Ensemble IDs I suggest you convert to gene symbols first, then sort by name (in excel) to identify any duplicates which you can then remove. If you're still having trouble, email me: katherine.west at glasgow.ac.uk and I'll have a look at your file
@@GenomicsGurus I do not have any duplicates in the list. The error I get is "After pruning, none of the gene sets passed size threshold". I can send you the screenshot and the file.
Ok, send the file and screenshot and I'll have a look.
Hello, It is really a great video. I have a question related to validation of repurposed drugs using GSEA. Actually, I am a beginner and want to do this. Can you please guide me how can I validate the drugs that I have using GSEA? Looking forward for your response
@Genomics Gurus
very helpful, thanks!
Glad it was helpful!
Extremlly useful, great presentation! How about ssGSEA?
Don't know about that one - sorry!
@@GenomicsGurus I just performed GSEA it is working perfectly! Thank you again...
Excellent - well done. Hope you got some interesting results!
Thank you so much!
Thank you so much, well done 🌹✨✔👌
Glad you found it useful!
Brilliant ! Big thank :)
Glad you liked it!
Nice video, very useful!
Many thanks Furong. Pleased you found it useful!
A great help. Thankyou mam
THANK YOU!!!!
Great video!
Thanks a lot. Glad you found it useful!
Firstly, thank you for the tutorial, I have found it really helpful.
I have a question regarding to what can be concluded from the enrichment. I have performed a preranked method and the results showed an enrichment in the pathway, however one of the genes that is highly expressed has an inhibitory function in the pathway. So my question is if the ES shows only the enrichment of the genes (either activators or inhibitors) or also the directionality of the pathway.
Thank you in advance.
Think it only tells you about the pathway in general and not on the individual genes in the pathway. In case you want to know about the directionality for individual genes, probably you would have to check your individual genes and the fold change for that gene from your DE table.
Amazing tutorial.
My question is, if i have RNA-seq results giving different expressed genes for different cell lines and I want to compare in order to check for example which cell line expresses genes (significant upregulated enrichment) associated with angiogenesis. In this case, in my expression dataset file, the first column would include ENS id's for these genes, but I have sometimes completely different genes in different cell lines (with 3 samples per cell line), so what is the appropriate way to organize this data?
comfortable voice
Great video! Thanks a lot for the explanation. I was wondering if you know how to use a continuous phenotype label for a time course actually. I tried following the user guide for that but I couldn't really do it. Thanks :)
I've not tried it myself, but it doesn't look too complicated. There are lots of ways to make mistakes with these input files, though, as my students will testify! If you want to email your files to me, I can have a look at them.....katherine.west at glasgow.ac.uk
This content is a treasure. Thank you so much Dr. West.
Just in case someone reads my comment: I have doubts about which values I need to put in the Expression data set file (10:37). I just have two groups and I have the "Raw comparison" values and other corrected values such as reads per kilobase per million (RPKM) and Transcripts Per Kilobase Million (TPM). Which one should I use?
Another question: My data comes from a RNA-seq analysis of Mus musculus cells. Which Chip platform (23:40) should I choose?
Many thanks! : )
Hi Joan, sorry for the slow reply. TPM is probably the best dataset to use. Your genes are probably named as ensemble gene IDs (ENSMUSGXxxx) so "mouse ensembl gene ID human orthologs" with the latest number would be the right chip platform to use.
@@GenomicsGurus Many Many thanks! : )
Hi Dr. West, great tutorial. I have one question looking for your help. When creating Phenotype labels file in excel, you mentioned to save as tab-delimited text file with the extension.cls. How to do this? Excel does not have an option to save a file as cls file. Thanks.
Hi, type yourfilename.cls in the file name box then save as tab delimited text. It will save as yourfilename.cls.txt , which will work. good luck!
Thank you very much for your tutorial. I have one question: Have you previously filtered the expression data? I mean, for example, applying a given Fold Change and filtering it by P-value.
Sorry for the slow reply. No, for GSEA you should use the whole data set, do not filter it.
Good stuff, thanks!
Glad you liked it!
Excellent tutorial and this made my concepts so clear. Just wondering if I can use GSEA for RNA seq analysis of any other organism. I am struggling with some Mycobacterium tuberculosis RNA seq data and could not find the GMT files for it. Is there any database from where I can download the GMT files for Mycobacterium tuberculosis.
I'm not sure if there are M. tuberculosis databases out there, but it's easy enough to make your own GMT files - it's just a list of gene IDs with a couple of rows at the top. You can download any current GMT file to see the format. I suggest you use the literature to find the genes associated with the pathway/phenotype you are interested in and make your own list. Good luck!
@@GenomicsGurus Thanks
Can you please make a video about how to make GCT file, thanks in advance :)
Hi, thanks for the tutorial. I had some trouble downloading the GSEA software so I ended up using the GenePattern UI. Is there any downside to using this website instead of the GSEA desktop application? Thanks
Thank you so much for your tutorial! In case I don't have the gene ID, just the gene symbol, how can I find their respective ID?
You shouldn't need the gene ID - choose a gene symbol chip platform instead of a gene ID chip platform when running GSEA. The long answer to your question is that you can download a file from ensembl that lists gene ID and gene symbols, and you can use vlookup in excel to look up IDs for known gene symbols and vice versa.