Hello,

I have a question about TMM normalization used in EdgeR.

Q. How can I get the normalized read counts from TMM?

I understand that calcNormFactors() produces two columns of information. The first is lib.size and the second is norm.factors. From what I have read, multiplying these two columns together gives an effective library size. Should I then be dividing the raw read counts by the effective library size to get normalized read counts? I am trying to do differential expression analysis on paired data using Fisher's exact test. By paired I mean I have two sets of data, sequenced on different platforms, but from the same patient. So I am looking for DEG caused by platform difference. Originally I was using RPKM values, but I am wondering if TMM would be better.


The article Normalization methods for Illumina high-throughput RNA sequencing data analysis describes normalized read counts in the following way.
"To obtain normalized read counts, these normalization factors are re-scaled by the mean of the normalized library sizes. Normalized read counts are obtained by dividing raw read counts by these re-scaled normalization factors."

I would love some clarification of TMM as well as any opinions on my use of Fisher's exact test. Thanks for the help in advance.

Reanne Bowlby

	[[alternative HTML version deleted]]

