Question: RNA-seq normalization and standardization
0
gravatar for hvphan
15 months ago by
hvphan0
hvphan0 wrote:

Suppose I have a matrix of gene counts, whose rows represent genes, and columns represent samples. For RNA-seq packages such as edgeR and DEseq2, the counts are normalized and standardized before DE analysis.

My question is about the standardization itself. After normalizing each column to adjust for sequencing depth and library size, I calculate the log2(normalized counts + 1). Then, is each column further standardized (to get mean = 0 and standard deviation = 1), or is the standardization done on each row only?

Thank you for your help.

rna-seq • 943 views
ADD COMMENTlink written 15 months ago by hvphan0

For RNA-seq packages such as edgeR and DEseq2, the counts are normalized and standardized before DE analysis.

You don't have to do anything to the counts, just give them raw to DESeq2/edgeR.

ADD REPLYlink written 15 months ago by WouterDeCoster42k

No need of standardization, just give raw data. And, Deseq2 won't accept even your normalized data.

ADD REPLYlink written 15 months ago by k.kathirvel93210

I just want to understand what the packages do to the raw counts in the background.

Do these packages calculate a normalization factor (like what Damian Kao said), scale the counts by the factor and do DE test? Do they standardize the normalized counts before DE test?

ADD REPLYlink written 15 months ago by hvphan0
1

Then you should probably read the paper and vignette

ADD REPLYlink written 15 months ago by WouterDeCoster42k
0
gravatar for Damian Kao
15 months ago by
Damian Kao15k
USA
Damian Kao15k wrote:

Packages like edgeR and DESeq2 don't actually transform the counts for DE analysis. A normalization factor is calculated and then used in the DE test. There are good information in the absolute counts for each gene. Standardization is not used, otherwise that information would be lost.

ADD COMMENTlink written 15 months ago by Damian Kao15k
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