tilingArray (version 1.50.0)

normalizeByReference: Probe-specific normalization of hybridization intensities from an oligonucleotide microarray

Description

Adjust the hybridization intensities from an oligonucleotide microarray for probe-specific response effect by using one or several reference hybridizations. If x contains more than one array, vsnMatrix from the vsn package is called for between array normalization.

Usage

normalizeByReference(x, reference, pm, background, refSig, nrStrata=10, cutoffQuantile=0.05, plotFileNames, verbose=FALSE)

Arguments

x

ExpressionSet containing the data to be normalized.

reference

ExpressionSet with the same number of features as x, containing the reference signal, on the raw scale (non-logarithmic). This argument can be used to directly input the data from a set of replicate DNA hybridizations. Alternatively, the argument refSig can be specified.

pm

Indices specifying the perfect match features in reference (see Details). This can be either an integer vector with values between 1 and nrow(exprs(reference)) or a logical vector.

background

Indices specifying a set of background features in x (see Details). This can be either an integer vector with values between 1 and nrow(exprs(x)) or a logical vector.

refSig

A numeric vector of the same length as pm with estimates of probe response effects, on a logarithm-like scale. This argument can be specified alternatively to reference.

nrStrata

Integer (length 1), number of strata for the estimation of the background function.

cutoffQuantile

Numeric (length 1), the probes whose reference signal is below this quantile are thrown out.

plotFileNames

Character vector whose length is the same as the number of arrays in x. Optional, if missing, no plots are produced.

verbose

Logical of length 1, if TRUE, some messages about progress are printed.

Value

A copy of x with the normalized intensities.

Details

The intensities in x are adjusted according to the reference values. Typically, the reference values are obtained by hybridizing a DNA sample to the array, so that the abundance of target is the same for all reference probes, and their signal can be used to estimate the probe sequence effect. A reference probe is a probe that perfectly matches the target genome exactly once. Usually, not all probes on a chip are reference probes, hence the subset of those that are is specified by the argument pm.

The background signal is estimated from the probes indicated by the argument background. They need to be a strict subset of the reference probes. I.e., they need to uniquely match the target organism's DNA, but are not expected to match any of its transcripts. A robust estimation method is used, so a small fraction of background probes that do hit transcripts is not harmful.

A limitation of this normalization method is that it only makes sense for the data from reference probes, NA values are returned for all other probes.

The functions PMindex and BGindex can be used to produce the pm and background arguments from a probeAnno environment such as provided in the davidTiling package.

To summarize, a reference probe (indicated by argument pm) is a probe that perfectly matches the target genome exactly once, a background probe (indicated by argument background) is a reference probe which we expect not to be transcribed. These should not be confused with what is called 'perfect match' and 'mismatch' probes in Affymetrix annotation.

References

The method implemented in this function is described in detail in Section 2.3 of the article Huber W, Toedling J, Steinmetz, L. Transcript mapping with high-density oligonucleotide tiling arrays. Bioinformatics 22, 1963-1970 (2006).

See Also

PMindex, BGindex

Examples

  ## see vignette assessNorm.Rnw in inst/scripts directory