diff --git a/README.md b/README.md index 63f37776ff98ca4ae325559638955f9151b49ebf..ecf461c22e4fda406131257b2ace1d4e6509382e 100644 --- a/README.md +++ b/README.md @@ -8,20 +8,39 @@ Purpose: ## About DESEQ2 +Main step: + +1) Estimate size factor + +Median ratio method is used to estimate the size factor per sample. + +The size factor is used for normalizing counts (per gene per sample). +Normalized counts allow minimizing biais linked to library size. +By normalizing the counts DESEQ2 aims to make sure differential expression are based on factors study and not to sequencing depth +/!\ gene length is not take into account ! + +2) Estimate dispersion + +Purpose: Estimate the variability between replicates + +Get dispersion estimate for each gene using Maximum Linkelihood Estimatation +Fit a curve to wise gene dispersion estimate + +3) Fit linear model + The differential expression analysis uses a generalized linear model of the form: -Kij ∼ NB(µij , α i ) -µij = s j q ij -log 2 (q ij ) = x j. β i +Kij ∼ NB(µij , α i )<br/> +µij = s j q ij <br/> +log 2 (q ij ) = x j. β i <br/> where counts K ij for gene i, sample j are modeled using a Negative Binomial distribution with fitted mean µ ij and a gene-specific dispersion parameter α i . The fitted mean is composed of a sample-specific size factor s j and a parameter q ij proportional to the expected true concentration of fragments for sample j. The coefficients β i give the log2 fold changes for gene i for each col- -umn of the model matrix X. The sample-specific size factors can be replaced by gene-specific -normalization factors for each sample using normalizationFactors. +umn of the model matrix X. The sample-specific size factors can be see as the logFC between condition. + +4) Wald Test H0: Log(FC) = 0 -Experiments without replicates do not allow for estimation of the dispersion of counts around the -expected value for each group, which is critical for differential expression analysis. Analysis with-out replicates is no longer supported since v1.22. ## Investigation of µ effect