normalizeByReference {tilingArray} | R Documentation

## 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.

### 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.

### Value

A copy of `x` with the normalized intensities.

### Author(s)

W. Huber huber@ebi.ac.uk

### 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

[Package tilingArray version 1.64.0 Index]