Question: Deterimining overall number of differentially expressed genes between WT and KO samples
0
gravatar for singcell
5 weeks ago by
singcell0
singcell0 wrote:

I am comparing differentially expressed genes (DEGs) between WT and KO samples. If I want to compare DEGs between conditions in a particular cluster I use FindMarkers function of Seurat tool by indicating ident.1 and ident.2.

However if I wanted to compare overall DEGs between WT and KO samples and NOT specific to any cluster. How can I accomplish in Seurat or using other tool?

Thank you,

ADD COMMENTlink modified 5 weeks ago • written 5 weeks ago by singcell0

Hi,

From what I read in the documentation of Seurat, if you set both ident.1 and ident.2 parameters to NULL (the default by the way) it will do (according to the docs): if NULL, use all other cells for comparison. See also the explanation on the Seurat vignettes/tutorials: If the ident.2 parameter is omitted or set to NULL, FindMarkers will test for differentially expressed features between the group specified by ident.1 and all other cells

So, try something like:

deg_ko_vs_wt <- FindMarkers(seurat_obj, ident.1 = NULL, ident.2 = NULL)

I hope this answers your question.

António

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by antonioggsousa910
1

Sorry, this will never work because you are comparing everything against everything, so obviously it'll not work. It only works when you want to compare one group against everything, then you can set the identity for that group ident.1 = group and the second identity group as NULL - ident.2 = NULL

Sorry by the mistake.

Follow the solution provided by @jomo018

António

ADD REPLYlink written 5 weeks ago by antonioggsousa910

@jomo018 way worked. Thank you for your help, anyway.

ADD REPLYlink written 5 weeks ago by singcell0

@jomo018 way worked. Thank you for your help, anyway.

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by singcell0
2
gravatar for jomo018
5 weeks ago by
jomo018610
jomo018610 wrote:

You need to define ident.1 in FindMarkers and cannot keep it as NULL.

Allowed idents are members of the named vector Idents(seurat_obj). The names of this vector are cell names. Originally, Idents(seurat_obj) holds cluster indices which lends itself to find markers between clusters.

However you can set Idents(seurat_obj) to other variables.

An easy way to do this is by using one of the columns in the metadata matrix of seurat_obj (whose rownames are also cell names). Check head(seurat_obj@meta.data) to determine if you already have a column holding KO or WT per each row. Say this column name is stim. You can now redefine Idents as follows: Idents(seurat_obj) <- seurat_obj$stim and continue with

FindMarkers(seurat_obj, ident.1="KO" ident.2="WT").

If you do not have a proper column in the metadata matrix, create a new one which holds KO/WT information (e.g. seurat_obj$newColumn<-...) and then use this column to redefine Idents.

ADD COMMENTlink modified 5 weeks ago • written 5 weeks ago by jomo018610

With ident.1="KO" and ident.2="WT", positive avg_logFC from FindMarkers indicates over-expression of KO relative to WT.

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by jomo018610

@singcell - it is probably better to convert your response from answer to comment. Otherwise, please provide more details about the problem that you actually see.

ADD REPLYlink written 5 weeks ago by jomo018610

Thank you for suggestion. I am new to this website. How can I convert my response from answer to comment?

ADD REPLYlink written 5 weeks ago by singcell0
0
gravatar for singcell
5 weeks ago by
singcell0
singcell0 wrote:

@jomo018 Thank you for your help. It worked however, I am seeing the FC other way round. When I run Idents(seurat_obj) I see multiple levels or clusters. I am wondering how Idents(seurat_obj) <- seurat_obj$stimis able to assign cells to correct idents or KO or WT.

ADD COMMENTlink written 5 weeks ago by singcell0
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