limmaCtData {HTqPCR}R Documentation

Differentially expressed features with qPCR: limma

Description

Function for detecting differentially expressed genes from high-throughput qPCR Ct values, based on the framework from the limma package. Multiple comparisons can be performed, and across more than two groups of samples.

Usage

limmaCtData(q, design = NULL, contrasts, sort = TRUE, stringent = TRUE, ndups = 1, spacing = NULL, dupcor, ...)

Arguments

q

object of class qPCRset.

design

matrix, design of the experiment rows corresponding to cards and columns to coefficients to be estimated. See details.

contrasts

matrix, with columns containing contrasts. See details

sort

boolean, should the output be sorted by adjusted p-values.

stringent

boolean, for flagging results as "Undetermined". See details.

ndups

integer, the number of times each feature is present on the card.

spacing

integer, the spacing between duplicate spots, spacing=1 for consecutive spots

dupcor

list, the output from duplicateCorrelation. See details.

...

any other arguments are passed to lmFit, contrasts.fit, eBayes or decideTests.

Details

This function is a wrapper for the functions lmFit, contrasts.fit (if a contrast matrix is supplied) and eBayes from the limma package. See the help pages for these functions for more information about setting up the design and contrast matrices.

All results are assigned to a category, either "OK" or "Unreliable" depending on the input Ct values. If stringent=TRUE any unreliable or undetermined measurements among technical and biological replicates will result in the final result being "Undetermined". For stringent=FALSE the result will be "OK" unless at least half of the Ct values for a given gene are unreliable/undetermined.

Note that when there are replicated features in the samples, each feature is assumed to be present the same number of times, and with regular spacing between replicates. Reordering the sample by featureNames and setting spacing=1 is recommendable.

If technical sample replicates are available, dupcor can be used. It is a list containing the estimated correlation between replicates. limmaCtData will then take this correlation into account when fitting a model for each gene. It can be calculate using the function duplicateCorrelation. Technical replicates and duplicated spots can't be assessed at the same time though, so if dupcor is used, ndups should be 1.

Value

A list of data.frames, one for each column in design, or for each comparison in contrasts if this matrix is supplied. Each component of the list contains the result of the given comparisons, with one row per gene and has the columns:

genes

Feature IDs.

feature.pos

The unique feature IDs from featurePos of the q object. Useful if replicates are not collapsed, in which case there might be several features with identical names.

t.test

The result of the t-test.

p.value

The corresponding p.values.

adj.p.value

P-values after correcting for multiple testing using the Benjamini-Holm method.

ddCt

The deltadeltaCt values.

FC

The fold change; 2^(-ddCt).

meanTest

The average Ct across the test samples for the given comparison.

meanReference

The average Ct across the reference samples for the given comparison.

categoryTest

The category of the Ct values ("OK", "Undetermined") across the test samples for the given comparison.

categoryReference

The category of the Ct values ("OK", "Undetermined") across the reference samples for the given comparison.

Also, the last item in the list is called "Summary", and it's the result of calling decideTests from limma on the fitted data. This is a data frame with one row per feature and one column per comparison, with down-regulation, no change and up-regulation marked by -1, 0 and 1.

Author(s)

Heidi Dvinge

References

Smyth, G. K. (2005). Limma: linear models for microarray data. In: Bioinformatics and Computational Biology Solutions using R and Bioconductor. R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, W. Huber (eds), Springer, New York, pages 397–420.

See Also

lmFit, contrasts.fit and ebayes for more information about the underlying limma functions. mannwhitneyCtData and ttestCtData for other functions calculating differential expression of Ct data. plotCtRQ, heatmapSig and plotCtSignificance can be used for visualising the results.

Examples

# Load example preprocessed data
data(qPCRpros)
samples <- read.delim(file.path(system.file("exData", package="HTqPCR"), "files.txt"))
# Define design and contrasts
design <- model.matrix(~0+samples$Treatment)
colnames(design) <- c("Control", "LongStarve", "Starve")
contrasts <- makeContrasts(LongStarve-Control, LongStarve-Starve, Starve-Control, levels=design)
# The actual test
diff.exp <- limmaCtData(qPCRpros, design=design, contrasts=contrasts)
# Some of the results
diff.exp[["LongStarve - Control"]][1:10,]

[Package HTqPCR version 1.40.0 Index]