- estimation of size factors:
`estimateSizeFactors`

- estimation of dispersion:
`estimateDispersions`

- Negative Binomial GLM fitting and Wald statistics:
`nbinomWaldTest`

`DESeq`

function returns a DESeqDataSet object,
results tables (log2 fold changes and p-values) can be generated
using the `results`

function. See the manual page
for `results`

for information on independent filtering and
p-value adjustment for multiple test correction.```
DESeq(object, test = c("Wald", "LRT"), fitType = c("parametric", "local",
"mean"), betaPrior, full = design(object), reduced, quiet = FALSE,
minReplicatesForReplace = 7, modelMatrixType, parallel = FALSE,
BPPARAM = bpparam())
```

object

a DESeqDataSet object, see the constructor functions

`DESeqDataSet`

,
`DESeqDataSetFromMatrix`

,
`DESeqDataSetFromHTSeqCount`

.test

either "Wald" or "LRT", which will then use either
Wald significance tests (defined by

`nbinomWaldTest`

),
or the likelihood ratio test on the difference in deviance between a
full and reduced model formula (defined by `nbinomLRT`

)fitType

either "parametric", "local", or "mean"
for the type of fitting of dispersions to the mean intensity.
See

`estimateDispersions`

for description.betaPrior

whether or not to put a zero-mean normal prior on
the non-intercept coefficients
See

`nbinomWaldTest`

for description of the calculation
of the beta prior. By default, the beta prior is used only for the
Wald test, but can also be specified for the likelihood ratio test.full

for

`test="LRT"`

, the full model formula,
which is restricted to the formula in `design(object)`

.
alternatively, it can be a model matrix constructed by the user.
advanced use: specifying a model matrix for full and `test="Wald"`

is possible if `betaPrior=FALSE`

reduced

for

`test="LRT"`

, a reduced formula to compare against,
i.e., the full formula with the term(s) of interest removed.
alternatively, it can be a model matrix constructed by the userquiet

whether to print messages at each step

minReplicatesForReplace

the minimum number of replicates required
in order to use

`replaceOutliers`

on a
sample. If there are samples with so many replicates, the model will
be refit after these replacing outliers, flagged by Cook's distance.
Set to `Inf`

in order to never replace outliers.modelMatrixType

either "standard" or "expanded", which describe
how the model matrix, X of the GLM formula is formed.
"standard" is as created by

`model.matrix`

using the
design formula. "expanded" includes an indicator variable for each
level of factors in addition to an intercept. for more information
see the Description of `nbinomWaldTest`

.
betaPrior must be set to TRUE in order for expanded model matrices
to be fit.parallel

if FALSE, no parallelization. if TRUE, parallel
execution using

`BiocParallel`

, see next argument `BPPARAM`

.
A note on running in parallel using `BiocParallel`

: it may be
advantageous to remove large, unneeded objects from your current
R environment before calling `DESeq`

,
as it is possible that R's internal garbage collection
will copy these files while running on worker nodes.- a
`DESeqDataSet`

object with results stored as metadata columns. These results should accessed by calling the`results`

function. By default this will return the log2 fold changes and p-values for the last variable in the design formula. See`results`

for how to access results for other variables.

$$K_{ij} \sim \textrm{NB}( \mu_{ij}, \alpha_i)$$ $$\mu_{ij} = s_j q_{ij}$$ $$\log_2(q_{ij}) = x_{j.} \beta_i$$

where counts $K_{ij}$ for gene i, sample j are modeled using
a Negative Binomial distribution with fitted mean $\mu_{ij}$
and a gene-specific dispersion parameter $\alpha_i$.
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 $\beta_i$ give the log2 fold changes for gene i for each
column of the model matrix $X$.
The sample-specific size factors can be replaced by
gene-specific normalization factors for each sample using
`normalizationFactors`

.

For details on the fitting of the log2 fold changes and calculation of p-values,
see `nbinomWaldTest`

if using `test="Wald"`

,
or `nbinomLRT`

if using `test="LRT"`

.

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. If an experimental design is
supplied which does not contain the necessary degrees of freedom for differential
analysis, `DESeq`

will provide a message to the user and follow
the strategy outlined in Anders and Huber (2010)
under the section 'Working without replicates', wherein all the samples
are considered as replicates of a single group for the estimation of dispersion.
As noted in the reference above: "Some overestimation of the variance
may be expected, which will make that approach conservative."
Furthermore, "while one may not want to draw strong conclusions from such an analysis,
it may still be useful for exploration and hypothesis generation."

The argument `minReplicatesForReplace`

is used to decide which samples
are eligible for automatic replacement in the case of extreme Cook's distance.
By default, `DESeq`

will replace outliers if the Cook's distance is
large for a sample which has 7 or more replicates (including itself).
This replacement is performed by the `replaceOutliers`

function. This default behavior helps to prevent filtering genes
based on Cook's distance when there are many degrees of freedom.
See `results`

for more information about filtering using
Cook's distance, and the 'Dealing with outliers' section of the vignette.
Unlike the behavior of `replaceOutliers`

, here original counts are
kept in the matrix returned by `counts`

, original Cook's
distances are kept in `assays(dds)[["cooks"]]`

, and the replacement
counts used for fitting are kept in `assays(dds)[["replaceCounts"]]`

.

Note that if a log2 fold change prior is used (betaPrior=TRUE)
then expanded model matrices will be used in fitting. These are
described in `nbinomWaldTest`

and in the vignette. The
`contrast`

argument of `results`

should be used for
generating results tables.

`nbinomWaldTest`

, `nbinomLRT`

```
# see vignette for suggestions on generating
# count tables from RNA-Seq data
cnts <- matrix(rnbinom(n=1000, mu=100, size=1/0.5), ncol=10)
cond <- factor(rep(1:2, each=5))
# object construction
dds <- DESeqDataSetFromMatrix(cnts, DataFrame(cond), ~ cond)
# standard analysis
dds <- DESeq(dds)
res <- results(dds)
# an alternate analysis: likelihood ratio test
ddsLRT <- DESeq(dds, test="LRT", reduced= ~ 1)
resLRT <- results(ddsLRT)
```

Run the code above in your browser using DataCamp Workspace