Volcano plot from a RNAseq excel file
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3 months ago

Hello everyone, I am new here and new to BioInformatic analysis. I need some help, please. Currently, I have downloaded an RNAseq file from Cibioportal. The file was then pasted into Excel. So I have the patient IDs and different gene expression values. I want to pick a gene and group it into two groups (high and low). And based on these two groups, generate a VOLCANOPLOT and see which genes are up or down-regulated by following my gene expression. Thank you very much for your help.

Volcanoplot • 644 views
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The file was then pasted into Excel.

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Hi, In fact, I've download "R" and even an "EnhancedVolcano" package. It's a little tricky to me. For example, how to specify the file in R. Does R generate automatically the Fold2change and the adjust p-value or I have to calculate them on my Own.

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Follow this tutorial for EnhancedVolcano: https://bioconductor.org/packages/release/bioc/vignettes/EnhancedVolcano/inst/doc/EnhancedVolcano.html

Does R generate automatically the Fold2change and the adjust p-value or I have to calculate them on my Own.

If the data is not analyzed then you will need to use a package such as DESeq2 BEFORE you try to use EnhancedVolcano. Tutorial for DESeq2 is here: http://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html

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Thank you very much. I'm going to try this

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I am really trying to understand all these procedures. I am a beginner in this field so please excuse my obvious questions. Actually, the RNAseq file from Cbioportal has the RSEM format. All I want to do is generate Fold2change and adjust the p-value to create the volcano graph. I am wondering how to import the file (RSEM) and perform the 2 analyses (fold change and adjust p value/ volcano plot).

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RSEM is a tool that generates expected counts, FPKM and TPM metrics for samples from their RNAseq raw data files. You may want to use the expected counts column.

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You don't "just" generate fold changes and p-values. You use special software that understands the nature of count data to calculate that for you. Given what you've got I strongly recommend using the tximport library to import the RSEM files into R, and DESeq2 can take it from there.

I also strongly recommend that you put your own data aside for a few days, and work through at last two differnt DESeq2 tutorials. It is not at all easy to learn how to use R while learning to use DESeq, but if that's what you have to do, then you need to sit down and do it, and not rush and process your own data wrong.

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Thank you for your replies. I'm going to test this procedure.