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Reanne Bowlby
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@reanne-bowlby-5546
Last seen 7.3 years ago

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
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