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LimmaNormalizer

Normalizes two-channel and single-channel microarrays using the Bioconductor Limma package. The inputs are raw green and red channel intensities and their corresponding background estimates. NA values are allowed. The output are normalized log-ratios; NA values may be present. All input matrices must have the same number of columns, but their column names do not need to be equal. Red inputs and several parameters are not used with single-channel data.

Many of the parameters correspond to Limma function parameters. Refer to Limma documentation for details. The component uses the version of Limma that is installed, but the list of valid parameter values in this documentation are obtained from Limma 3.6.9.

Note that for single-channel, no within-array normalization is done. For two-channel between-array normalization is usually not required.

For Agilent arrays, suggested source columns for green, greenBG, red and redBG are gMedianSignal, gBGMedianSignal, rMedianSignal and rBGMedianSignal.

Version 0.6
Bundle microarray
Categories Data Import Normalization
Authors Kristian Ovaska (kristian.ovaska@helsinki.fi), Viljami Aittomaki (viljami.aittomaki@helsinki.fi)
Issue tracker View/Report issues
Requires limma (R-bioconductor)
Source files component.xml LimmaNormalizer.r
Usage Example with default values

Inputs

Name Type Mandatory Description
green LogMatrix Mandatory Raw (non-normalized) green channel foreground intensities.
greenBG LogMatrix Mandatory Green channel background intensities.
red LogMatrix Optional Raw (non-normalized) red channel foreground intensities. Not used for single-channel data.
redBG LogMatrix Optional Red channel background intensities. Not used for single-channel data.
groups SampleGroupTable Optional If defined, names of output log-ratio columns and the directions of each log-ratio group are determined from here. For each green/red column pair G/R, there should be an entry of type ratio having the columns G and R as members. If the members are in order G,R (green channel first), the log-ratio for this group is G-R; otherwise, it is R-G. If the input is omitted, redMinusGreen parameter determines the direction and outColumnPattern determines the output column names. Not used for single-channel data.

Outputs

Name Type Description
logratio LogMatrix Normalized log-ratios. The i'th column corresponds to the i'th column in the input matrices.

Parameters

Name Type Default Description
arrayMethod string "none" Method for normalizing between arrays. Corresponds to the method parameter of Limma normalizeBetweenArrays function. Valid values: none, scale, quantile, Aquantile, Gquantile, Rquantile or cyclicloess (where the Xquantile options are not valid for single-channel).
backgroundMethod string "normexp" Method for background correction. Corresponds to the method parameter of Limma backgroundCorrect function. Valid values: none, subtract, half, minimum, movingmin, edwards, normexp.
loessIterations int 4 For LOESS normalization, controls the number of iterations. Corresponds to the iterations parameter of Limma normalizeWithinArrays function. Not used for single-channel data.
loessSpan float 0.3 For LOESS normalization, controls the smoothing of the LOESS curve. Corresponds to the span parameter of Limma normalizeWithinArrays function. Not used for single-channel data.
logratioMethod string "loess" Method for normalizing log-ratios to account for dyes and other factors. Corresponds to the method parameter of Limma normalizeWithinArrays function. Valid values: none, median, loess. Not used for single-channel data.
normexpMethod string "mle" If backgroundMethod = normexp, this is an additional parameter for background estimation. Valid values: saddle, mle, rma (needs Bioconductor affy package), rma75.
outColumnPattern string "%s_%s" If the sample groups are not defined, this gives the pattern for naming log-ratio output columns. If redMinusGreen is true, the first %s is replaced with the red channel name and the second %s with the green one. If redMinusGreen is false, column names are reversed. Not used for single-channel data.
redMinusGreen boolean true If the sample groups are not defined, this defines the direction of log-ratio computation. If true, all log-ratios are red minus green channel; if false, they are green minus red channel. Not used for single-channel data.

Test cases

Test case Parameters IN
green
IN
greenBG
IN
red
IN
redBG
IN
groups
OUT
logratio
case1_nonorm properties green greenBG red redBG groups logratio

backgroundMethod=none,
logratioMethod=none

case2_subtract properties green greenBG red redBG groups logratio

backgroundMethod=subtract,
logratioMethod=none

case3_subtract2 properties green greenBG red redBG (missing) logratio

backgroundMethod=subtract,
logratioMethod=none,
redMinusGreen=false

case4_defaults (missing) green greenBG red redBG (missing) logratio

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