estimateSizeFactors
Estimate the size factors for a DESeqDataSet
This function estimates the size factors using the
"median ratio method" described by Equation 5 in Anders and Huber (2010).
The estimated size factors can be accessed using the accessor function sizeFactors
.
Alternative library size estimators can also be supplied
using the assignment function sizeFactors<
.
Usage
"estimateSizeFactors"(object, type = c("ratio", "iterate"), locfunc = stats::median, geoMeans, controlGenes, normMatrix)
Arguments
 object
 a DESeqDataSet
 type
 either "ratio" or "iterate". "ratio" uses the standard median ratio method introduced in DESeq. The size factor is the median ratio of the sample over a pseudosample: for each gene, the geometric mean of all samples. "iterate" offers an alternative estimator, which can be used even when all genes contain a sample with a zero. This estimator iterates between estimating the dispersion with a design of ~1, and finding a size factor vector by numerically optimizing the likelihood of the ~1 model.
 locfunc
 a function to compute a location for a sample. By default, the
median is used. However, especially for low counts, the
shorth
function from the genefilter package may give better results.  geoMeans
 by default this is not provided and the geometric means of the counts are calculated within the function. A vector of geometric means from another count matrix can be provided for a "frozen" size factor calculation
 controlGenes
 optional, numeric or logical index vector specifying those genes to use for size factor estimation (e.g. housekeeping or spikein genes)
 normMatrix
 optional, a matrix of normalization factors which do not yet
control for library size. Note that this argument should not be used (and
will be ignored) if the
dds
object was created usingtximport
. In this case, the information inassays(dds)[["avgTxLength"]]
is automatically used to create appropriate normalization factors. ProvidingnormMatrix
will estimate size factors on the count matrix divided bynormMatrix
and store the product of the size factors andnormMatrix
asnormalizationFactors
. It is recommended to divide out the rowwise geometric mean ofnormMatrix
so the rows roughly are centered on 1.
Details
Typically, the function is called with the idiom:
dds < estimateSizeFactors(dds)
See DESeq
for a description of the use of size factors in the GLM.
One should call this function after DESeqDataSet
unless size factors are manually specified with sizeFactors
.
Alternatively, genespecific normalization factors for each sample can be provided using
normalizationFactors
which will always preempt sizeFactors
in calculations.
Internally, the function calls estimateSizeFactorsForMatrix
,
which provides more details on the calculation.
Value

The DESeqDataSet passed as parameters, with the size factors filled
in.
References
Reference for the median ratio method:
Simon Anders, Wolfgang Huber: Differential expression analysis for sequence count data. Genome Biology 2010, 11:106. http://dx.doi.org/10.1186/gb20101110r106
See Also
Examples
dds < makeExampleDESeqDataSet(n=1000, m=4)
dds < estimateSizeFactors(dds)
sizeFactors(dds)
dds < estimateSizeFactors(dds, controlGenes=1:200)
m < matrix(runif(1000 * 4, .5, 1.5), ncol=4)
dds < estimateSizeFactors(dds, normMatrix=m)
normalizationFactors(dds)[1:3,]
geoMeans < exp(rowMeans(log(counts(dds))))
dds < estimateSizeFactors(dds,geoMeans=geoMeans)
sizeFactors(dds)