A scaling normalization method for differential expression analysis of RNA-seq data - Genome Biology

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• the distribution of M values

• TMM normalization is a simple and effective method for estimating relative RNA production levels from RNA-seq data

• The TMM method estimates scale factors between samples that can be incorporated into currently used statistical methods for DE analysis.

• We have shown that normalization is required in situations where the underlying distribution of expressed transcripts between samples is markedly different

• In essence, both microarray and TMM normalization assume that the majority of genes, common to both samples, are not differentially expressed

• two RNA populations, A and B

• Scaling to library size as a form of normalization makes intuitive sense

• suppose every gene that is expressed in B is expressed in A with the same number of transcripts

• The distribution of M values for these technical replicates is concentrated around zero.

• The M versus A plot in Figure 1c illustrates that there exists a prominent set of genes with higher expression in liver