moderated estimation of fold change and dispersio

Moderated estimation of fold change and dispersio pdf#

blue circles = genes which have high gene-wise dispersion estimates and are hence labelled dispersion outliers and not shrunk toward the fitted trend line.The values are used in the statistical testing. blue dot = the final dispersion estimates shrunk from the gene-wise estimates towards the fitted estimates.red line = fitted estimates showing the dispersions' dependence on the mean.black dot = dispersion estimate for each gene as obtained by considering the information from each gene separately.Plot of dispersion estimates at different count levels, showing.MA scatter plot where the significantly differentially expressed genes are highlighted.

Moderated estimation of fold change and dispersio pdf#

deseq2_report.pdf: A PDF file containing:.

summary.txt: Textual summary of the differential expression results, including information on filtering and outliers.

de-list-deseq2.bed: The BED version of the results table contains genomic coordinates and log2 fold change values.

padj = Benjamini-Hochberg adjusted p-value.

lfcSE = standard error of the log2FoldChange estimate.

value 2 means that the expression has increased 4-fold

log2FoldChange = log2 fold change between the groups.

baseMean = the average of the normalized counts taken over all samples.

de-list-deseq2.tsv: Table containing the significantly differentially expressed genes.

Note that if you have more than two experimental groups, the output figures sum up information from all pairwise comparisons. The analysis output consists of the following files. Removing these genes with low counts improves the detection power by making the multiple testing adjustment of the p-values less severe. It also automatically removes genes whose mean of normalized counts is below a thresholdĭetermined by an optimization procedure. In addition to the group information, you can give an additional experimental factor like pairing to the analysis.ĭESeq2 detects automatically count outliers using Cooks's distance and removes these genes from analysis. If there are no replicates, DESeq will estimate dispersion using the samples from the different conditions as replicates.ĭESeq2 fits negative binomial generalized linear models for each gene and uses the Wald test for significance testing. You need to have biological replicates of each experiment condition in order to estimate dispersion properly. This procedureĬorrects for library size and RNA composition bias, which can arise for example when only a small number of genes are very highly expressed in one experiment condition but not in the other.Īs small numbers of replicates make it impossible to estimate within-group variance reliably, DESeq2 uses shrinkage estimation for dispersions and fold changes.Ī dispersion value is estimated for each gene through a model fit procedure. The median of these ratios in a sample is the size factor for that sample. The counts for a gene in each sample is then divided by this mean. These files are best created by the tool 'Utilities / Define NGS experiment', which combines count files for different samples to one table,ĭESeq2 performs an internal normalization where geometric mean is calculated for each gene across all samples. The count table has to be associated with a phenodata file describing the experimental groups. This tool takes as input a table of raw counts.

Cutoff for the adjusted P-value (0-1).

Column describing additional experimental factor.

This tool allows you to have more than two experimental groups andĪccount for a second experimental factor.

Differential expression analysis using DESeq2 Descriptionĭifferential expression analysis using the DESeq2 Bioconductor package.