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Question: threshold to filter lowly expressed genes
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gravatar for myprogramming2016
8 months ago by
myprogramming20160 wrote:

Hello,

I would like to use edgeR for differential Gene expression analysis. 

I have read counts data on 50 individuals in three biological replicates.

I would like to filter out lowly expressed genes. Is there a threshold to define express genes?  

I was thinking to use CPM of >=2, and it should be in two of the three libraries.

How do I define R code?

Secondly, I would like to output TMM normalized counts to identify uniquely expressed genes in each of the individuals. How do I output the TMM counts in CPM?

Thanks in advance

 

ADD COMMENTlink modified 8 months ago • written 8 months ago by myprogramming20160

Thanks for sharing an updated workflow. It is very helpful.

Secondly, I do not have a group (control Vs treated). I would like to identify differentially expressed genes in a particular individual compared with a mean of all the individuals (Intercept). I would like to repeat it for all the individuals.

I was wondering if there is any tutorial or an R script.

Thanks

 

ADD REPLYlink modified 8 months ago • written 8 months ago by myprogramming20160

Could you please reply to my post?

Thanks

ADD REPLYlink written 7 months ago by myprogramming20160
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gravatar for Gordon Smyth
8 months ago by
Gordon Smyth32k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth32k wrote:

The section called "Filtering to remove low counts" in the workflow article

   https://f1000research.com/articles/5-1438/v2

gives a clear rule of thumb for filtering that you could apply in your situation.

ADD COMMENTlink written 8 months ago by Gordon Smyth32k

I think that the current wording is confusing. The explanation states "This ensures that a gene will be retained if it is expressed in both the libraries belonging to any of the six groups.". I read this as the two samples will belong to the same class, whereas the filtering is independent of samples' classes and the two samples with sufficient reads could belong to two different classes.

ADD REPLYlink written 8 months ago by Dario Strbenac1.4k
1

The form of the quoted sentence is "if A then B", but you are reading it to mean "if B then A".

You're not the first person to make the same interpretation, so I guess that making the reasoning longer and even more explicit may have been worthwhile. I'm always surprised by how much confusion the issue of filtering causes. However we are not planning to make changes to this published article. Making changes would require the whole article to be re-refereed.

ADD REPLYlink modified 8 months ago • written 8 months ago by Gordon Smyth32k

Indeed, after reading it again, I noticed that I interpreted it incorrectly.

ADD REPLYlink written 8 months ago by Dario Strbenac1.4k
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