Question: differential expression volcano plot
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gravatar for michelle.piquet
5 weeks ago by
michelle.piquet10 wrote:

Hello,

I have generated the counts from 10x Chromium and then generated differential expression for two conditions, untreated and treated. I have done volcano plots for each cluster and then combined them all into one.

Is there a way to just do a volcano plot from the excel file all at once, rather than doing them one by one and then merging?

Thanks for any help and input.

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ADD COMMENTlink modified 5 weeks ago • written 5 weeks ago by michelle.piquet10

What have you tried? Can you point us to some of the manuals you've tried?

ADD REPLYlink written 5 weeks ago by RamRS25k

I have just loaded each count file into Seurat. Now, I'm trying to combine all treated and untreated samples and do differential expression and then do a volcano plot at the end.

ADD REPLYlink written 5 weeks ago by michelle.piquet10
1

Have you tried following any of the vignettes on the Satija Lab website? What effort have you invested in solving your own problem?

ADD REPLYlink written 5 weeks ago by RamRS25k

Yes. I have the csv file with the differential expression for both treated and untreated conditions. There are 20 total clusters, how do I go about merging the results table since all the p values and log fold change values are already in my file. They're just labeled based on which cluster it belongs to?

I used this code for 2 clusters...

v1_macrophage <- EnhancedVolcano(...)
v2_macrophage <- EnhancedVolcano(...)

grid.arrange(v1_macrophage, v2_macrophage, ncol = 2, top = textGrob('Macrophages', just = c('center'), gp = gpar(fontsize = 32)))

Is there a way to combine all the data from that one file and generate a volcano plot from that, rather than having to do one cluster at a time and then combining them?

These are the headers of the excel file:

Feature ID Feature Name Cluster 1 Mean Counts Cluster 1 Log2 fold change Cluster 1 Adjusted p value Cluster 2 Mean Counts Cluster 2 Log2 fold change Cluster 2 Adjusted p value Cluster 3 Mean Counts Cluster 3 Log2 fold change Cluster 3 Adjusted p value Cluster 4 Mean Counts Cluster 4 Log2 fold change Cluster 4 Adjusted p value Cluster 5 Mean Counts Cluster 5 Log2 fold change Cluster 5 Adjusted p value Cluster 6 Mean Counts Cluster 6 Log2 fold change Cluster 6 Adjusted p value Cluster 7 Mean Counts Cluster 7 Log2 fold change Cluster 7 Adjusted p value Cluster 8 Mean Counts Cluster 8 Log2 fold change Cluster 8 Adjusted p value Cluster 9 Mean Counts Cluster 9 Log2 fold change Cluster 9 Adjusted p value Cluster 10 Mean Counts Cluster 10 Log2 fold change Cluster 10 Adjusted p value Cluster 11 Mean Counts Cluster 11 Log2 fold change Cluster 11 Adjusted p value Cluster 12 Mean Counts Cluster 12 Log2 fold change Cluster 12 Adjusted p value Cluster 13 Mean Counts Cluster 13 Log2 fold change Cluster 13 Adjusted p value Cluster 14 Mean Counts Cluster 14 Log2 fold change Cluster 14 Adjusted p value Cluster 15 Mean Counts Cluster 15 Log2 fold change Cluster 15 Adjusted p value Cluster 16 Mean Counts Cluster 16 Log2 fold change Cluster 16 Adjusted p value Cluster 17 Mean Counts Cluster 17 Log2 fold change Cluster 17 Adjusted p value Cluster 18 Mean Counts Cluster 18 Log2 fold change Cluster 18 Adjusted p value Cluster 19 Mean Counts Cluster 19 Log2 fold change Cluster 19 Adjusted p value Cluster 20 Mean Counts Cluster 20 Log2 fold change Cluster 20 Adjusted p value
ADD REPLYlink modified 5 weeks ago by RamRS25k • written 5 weeks ago by michelle.piquet10

Is your question about automating EnhancedVolcano plot generation or analysis using Seurat?

ADD REPLYlink written 5 weeks ago by RamRS25k

It's both. I used Seurat to generate the differential expression file and now don't know how to combine all the information from the file into a volcanoplot within Seurat or with EnhancedVolcano, don't know which would allow me to merge all the information together.

I did the merge for 2 out of the 20 clusters by inputing the individuality, how would I got about just doing it as one script?

ADD REPLYlink written 5 weeks ago by michelle.piquet10

So do you want volcano plots showing your differential expression analysis between each set of clusters? Or just between the treated and untreated conditions?

I recommend reading this tutorial and editing your question as appropriate so that users have the information necessary to help you.

ADD REPLYlink written 5 weeks ago by jared.andrews074.3k

No, just between the treated and untreated conditions. I already have the differential expression analysis excel file, I'm just stuck on how to merge everything together. I know how to do a volcano plot for each cluster and then merge but can't seem to just merge everything into one and do the volcano plot.

ADD REPLYlink written 5 weeks ago by michelle.piquet10

Previous discussion: A: Volcano plot for multiple clusters

Michelle, you just need to bind respective columns together, i.e., in pseudocode:

rbind(
  data[,Cluster 1 Mean Counts, Cluster 1 Log2 fold change, Cluster 1 Adjusted p value],
  data[,Cluster 2 Mean Counts, Cluster 2 Log2 fold change, Cluster 2 Adjusted p value],
  ...
  data[,Cluster n Mean Counts, Cluster n Log2 fold change, Cluster n Adjusted p value])

Then generate a volcano from the final, merged, data-frame. You will have to do some work to force rbind() to bind columns with different names.

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by Kevin Blighe53k

Hi Kevin,

I don't understand what I'm suppose to do.

How do I go about binding everything in the file? I tried to change the names in the file to just log2foldchange and pvalue but when I upload the dataset, a 1-20 is added to the end of each.

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by michelle.piquet10

Then you need to do differential expression on your treatment conditions, not the clusters. Reading the Seurat reference manual documentation for FindAllMarkers and FindMarkers will show you how to do that.

ADD REPLYlink written 5 weeks ago by jared.andrews074.3k

I already did differential expression for both conditions, I just need to to plot them into a volcano plot.

ADD REPLYlink written 5 weeks ago by michelle.piquet10

What Jared says is, at the end of the day, what you likely want to do.

ADD REPLYlink written 5 weeks ago by Kevin Blighe53k

Okay, then what's your issue? Grab the log2 foldchanges and p-values for that comparison and go to town. You said you've been able to produce volcano plots, so it's not exactly clear where the sticking point is.

ADD REPLYlink written 5 weeks ago by jared.andrews074.3k
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