Can I use DESeq2 to analyze a dataset without replicates?.Can I run DESeq2 to contrast the levels of many groups?.If I have multiple groups, should I run all together or split into pairs of groups?.Can I use DESeq2 to analyze paired samples?.Do normalized counts correct for variables in the design?.Why after VST are there still batches in the PCA plot?.How do I use VST or rlog data for differential testing?.How can I get unfiltered DESeq2 results?.Independent filtering and multiple testing.Methods changes since the 2014 DESeq2 paper.Group-specific condition effects, individuals nested within groups.Sample-/gene-dependent normalization factors.Tests of log2 fold change above or below a threshold.Dispersion plot and fitting alternatives.Recommendations for single-cell analysis.

Extended section on shrinkage estimators.Control features for estimating size factors.Principal component plot of the samples.Heatmap of the sample-to-sample distances.Data quality assessment by sample clustering and visualization.Effects of transformations on the variance.Rich visualization and reporting of results.Log fold change shrinkage for visualization and ranking.Tximeta for import with automatic metadata.Transcript abundance files and tximport / tximeta.An RNA-seq workflow on the Bioconductor website covers similar material to this vignette but at a slower pace, including the generation of count matrices from FASTQ files. This vignette explains the use of the package and demonstrates typical workflows. The package DESeq2 provides methods to test for differential expression by use of negative binomial generalized linear models the estimates of dispersion and logarithmic fold changes incorporate data-driven prior distributions. An important analysis question is the quantification and statistical inference of systematic changes between conditions, as compared to within-condition variability. Analogous data also arise for other assay types, including comparative ChIP-Seq, HiC, shRNA screening, and mass spectrometry. The count data are presented as a table which reports, for each sample, the number of sequence fragments that have been assigned to each gene. A basic task in the analysis of count data from RNA-seq is the detection of differentially expressed genes.

Extened memory note 7