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DESeq2 employs the "size factor" normalization method, which adjusts for differences in sequencing depth between samples. [1] This normalization ensures that the expression values of genes are comparable across samples, allowing for accurate identification of differentially expressed genes.
By minimizing these systematic variations, true biological differences can be found. To determine whether normalization is needed, one can plot Cy5 (R) intensities against Cy3 (G) intensities and see whether the slope of the line is around 1. An improved method, which is basically a scaled, 45 degree rotation of the R vs. G plot is an MA-plot. [4]
cqn [35] is a normalization tool for RNA-Seq data, implementing the conditional quantile normalization method. EDASeq [36] is a Bioconductor package to perform GC-Content Normalization for RNA-Seq Data. GeneScissors A comprehensive approach to detecting and correcting spurious transcriptome inference due to RNAseq reads misalignment.
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Methods: Most tools use regression or non-parametric statistics to identify differentially expressed genes, and are either based on read counts mapped to a reference genome (DESeq2, limma, edgeR) or based on read counts derived from alignment-free quantification (sleuth, [106] Cuffdiff, [107] Ballgown [108]). [109]
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