Package 'limma'

Description

LIMMA is a package for the analysis of gene expression microarray data, especially the use of linear models for analysing designed experiments and the assessment of differential expression. LIMMA provides the ability to analyse comparisons between many RNA targets simultaneously in arbitrary complicated designed experiments. Empirical Bayesian methods are used to provide stable results even when the number of arrays is small. The linear model and differential expression functions apply to all gene expression technologies, including microarrays, RNA-seq and quantitative PCR.

Details

There are three types of documentation available:

1. The LIMMA User's Guide can be reached through the "User Guides and Package Vignettes" links at the top of the LIMMA contents page. The function limmaUsersGuide gives the file location of the User's Guide.
   

2. An overview of limma functions grouped by purpose is contained in the numbered chapters at the foot of the LIMMA package index page, of which this page is the first.
   

3. The LIMMA contents page gives an alphabetical index of detailed help topics.
   

The function changeLog displays the record of changes to the package.

Author(s)

Gordon Smyth, with contributions from many colleagues

References

Law CW, Chen Y, Shi W, Smyth GK (2014). Voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biology 15, R29. doi:10.1186/gb-2014-15-2-r29. See also the Preprint Version at https://gksmyth.github.io/pubs/VoomPreprint.pdf incorporating some notational corrections.

Phipson B, Lee S, Majewski IJ, Alexander WS, and Smyth GK (2016). Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Annals of Applied Statistics 10, 946-963. doi:10.1214/16-AOAS920

Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Research 43, e47. doi:10.1093/nar/gkv007

Smyth GK (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology Volume 3, Issue 1, Article 3. doi:10.2202/1544-6115.1027. See also the Preprint Version https://gksmyth.github.io/pubs/ebayes.pdf incorporating corrections to 30 June 2009.

See Also

02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

This package defines the following data classes.

RGList

A class used to store raw intensities as they are read in from an image analysis output file, usually by read.maimages.

MAList

Intensities converted to M-values and A-values, i.e., to with-spot and whole-spot contrasts on the log-scale. Usually created from an RGList using MA.RG or normalizeWithinArrays. Objects of this class contain one row for each spot. There may be more than one spot and therefore more than one row for each probe.

EListRaw

A class to store raw intensities for one-channel microarray data. May or may not be background corrected. Usually created by read.maimages.

EList

A class to store normalized log2 expression values for one-channel microarray data. Usually created by normalizeBetweenArrays.

MArrayLM

Store the result of fitting gene-wise linear models to the normalized intensities or log-ratios. Usually created by lmFit. Objects of this class normally contain only one row for each unique probe.

TestResults

Store the results of testing a set of contrasts equal to zero for each probe. Usually created by decideTests. Objects of this class normally contain one row for each unique probe.

All these data classes obey many analogies with matrices. In the case of RGList, MAList, EListRaw and EList, rows correspond to spots or probes and columns to arrays. In the case of MarrayLM, rows correspond to unique probes and the columns to parameters or contrasts. The functions summary, dim, length, ncol, nrow, dimnames, rownames, colnames have methods for these classes. Objects of any of these classes may be subsetted. Multiple data objects may be combined by rows (to add extra probes) or by columns (to add extra arrays).

Furthermore all of these classes may be coerced to actually be of class matrix using as.matrix, although this entails loss of information. Fitted model objects of class MArrayLM can be coerced to class data.frame using as.data.frame.

The first three classes belong to the virtual class LargeDataObject. A show method is defined for LargeDataOjects which uses the utility function printHead.

Author(s)

Gordon Smyth

See Also

01.Introduction, 02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

This help page gives an overview of LIMMA functions used to read data from files.

Reading Target Information

The function readTargets is designed to help with organizing information about which RNA sample is hybridized to each channel on each array and which files store information for each array.

Reading Intensity Data

The first step in a microarray data analysis is to read into R the intensity data for each array provided by an image analysis program. This is done using the function read.maimages.

read.maimages optionally constructs quality weights for each spot using quality functions listed in QualityWeights.

If the data is two-color, then read.maimages produces an RGList object. If the data is one-color (single channel) then an EListRaw object is produced. In either case, read.maimages stores only the information required from each image analysis output file. read.maimages uses utility functions removeExt, read.imagene and read.columns. There are also a series of utility functions which read the header information from image output files including readGPRHeader, readImaGeneHeader and readGenericHeader.

read.ilmn reads probe or gene summary profile files from Illumina BeadChips, and produces an ElistRaw object.

read.idat reads Illumina files in IDAT format, and produces an EListRaw object. detectionPValues can be used to add detection p-values.

The function as.MAList can be used to convert a marrayNorm object to an MAList object if the data was read and normalized using the marray and marrayNorm packages.

Reading the Gene List

Most image analysis software programs provide gene IDs as part of the intensity output files, for example GenePix, Imagene and the Stanford Microarray Database do this. In other cases the probe ID and annotation information may be in a separate file. The most common format for the probe annotation file is the GenePix Array List (GAL) file format. The function readGAL reads information from a GAL file and produces a data frame with standard column names.

The function getLayout extracts from the GAL-file data frame the print layout information for a spotted array. The functions gridr, gridc, spotr and spotc use the extracted layout to compute grid positions and spot positions within each grid for each spot. The function printorder calculates the printorder, plate number and plate row and column position for each spot given information about the printing process. The utility function getSpacing converts character strings specifying spacings of duplicate spots to numeric values.

The Australian Genome Research Facility in Australia often produces GAL files with composite probe IDs or names consisting of multiple strings separated by a delimiter. These can be separated into name and annotation information using strsplit2.

If each probe is printed more than once of the arrays in a regular pattern, then uniquegenelist will remove duplicate names from the gal-file or gene list.

Identifying Control Spots

The functions readSpotTypes and controlStatus assist with separating control spots from ordinary genes in the analysis and data exploration.

Manipulating Data Objects

cbind, rbind, merge allow different RGList or MAList objects to be combined. cbind combines data from different arrays assuming the layout of the arrays to be the same. merge can combine data even when the order of the probes on the arrays has changed. merge uses utility function makeUnique.

Author(s)

Gordon Smyth

See Also

01.Introduction, 02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

This page deals with background correction methods provided by the backgroundCorrect, kooperberg or neqc functions. Microarray data is typically background corrected by one of these functions before normalization and other downstream analysis.

backgroundCorrect works on matrices, EListRaw or RGList objects, and calls backgroundCorrect.matrix.

The movingmin method of backgroundCorrect uses utility functions ma3x3.matrix and ma3x3.spottedarray.

The normexp method of backgroundCorrect uses utility functions normexp.fit and normexp.signal.

kooperberg is a Bayesian background correction tool designed specifically for two-color GenePix data. It is computationally intensive and requires several additional columns from the GenePix data files. These can be read in using read.maimages and specifying the other.columns argument.

neqc is for single-color data. It performs normexp background correction and quantile normalization using control probes. It uses utility functions normexp.fit.control and normexp.signal. If robust=TRUE, then normexp.fit.control uses the function huber in the MASS package.

Author(s)

Gordon Smyth

See Also

01.Introduction, 02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

This page gives an overview of the LIMMA functions available to normalize data from single-channel or two-colour microarrays. Smyth and Speed (2003) give an overview of the normalization techniques implemented in the functions for two-colour arrays.

Usually data from spotted microarrays will be normalized using normalizeWithinArrays. A minority of data will also be normalized using normalizeBetweenArrays if diagnostic plots suggest a difference in scale between the arrays.

In rare circumstances, data might be normalized using normalizeForPrintorder before using normalizeWithinArrays.

All the normalization routines take account of spot quality weights which might be set in the data objects. The weights can be temporarily modified using modifyWeights to, for example, remove ratio control spots from the normalization process.

If one is planning analysis of single-channel information from the microarrays rather than analysis of differential expression based on log-ratios, then the data should be normalized using a single channel-normalization technique. Single channel normalization uses further options of the normalizeBetweenArrays function. For more details see the LIMMA User's Guide which includes a section on single-channel normalization.

normalizeWithinArrays uses utility functions MA.RG, loessFit and normalizeRobustSpline.

normalizeBetweenArrays is the main normalization function for one-channel arrays, as well as an optional function for two-colour arrays. normalizeBetweenArrays uses utility functions normalizeMedianValues, normalizeMedianAbsValues, normalizeQuantiles and normalizeCyclicLoess, none of which need to be called directly by users.

neqc is a between array normalization function customized for Illumina BeadChips.

The function normalizeVSN is also provided as a interface to the vsn package. It performs variance stabilizing normalization, an algorithm which includes background correction, within and between normalization together, and therefore doesn't fit into the paradigm of the other methods.

removeBatchEffect can be used to remove a batch effect, associated with hybridization time or some other technical variable, prior to unsupervised analysis.

Author(s)

Gordon Smyth

References

Smyth, G. K., and Speed, T. P. (2003). Normalization of cDNA microarray data. Methods 31, 265-273. https://gksmyth.github.io/pubs/normalize.pdf

See Also

01.Introduction, 02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

This page gives an overview of the LIMMA functions available to fit linear models and to interpret the results. This page covers models for two color arrays in terms of log-ratios or for single-channel arrays in terms of log-intensities. If you wish to fit models to the individual channel log-intensities from two colour arrays, see 07.SingleChannel.

The core of this package is the fitting of gene-wise linear models to microarray data. The basic idea is to estimate log-ratios between two or more target RNA samples simultaneously. See the LIMMA User's Guide for several case studies.

Fitting Models

The main function for model fitting is lmFit. This is recommended interface for most users. lmFit produces a fitted model object of class MArrayLM containing coefficients, standard errors and residual standard errors for each gene. lmFit calls one of the following three functions to do the actual computations:

lm.series

Straightforward least squares fitting of a linear model for each gene.

mrlm

An alternative to lm.series using robust regression as implemented by the rlm function in the MASS package.

gls.series

Generalized least squares taking into account correlations between duplicate spots (i.e., replicate spots on the same array) or related arrays. The function duplicateCorrelation is used to estimate the inter-duplicate or inter-block correlation before using gls.series.

All the functions which fit linear models use link{getEAW} to extract data from microarray data objects, and unwrapdups which provides an unified method for handling duplicate spots.

Forming the Design Matrix

lmFit has two main arguments, the expression data and the design matrix. The design matrix is essentially an indicator matrix which specifies which target RNA samples were applied to each channel on each array. There is considerable freedom in choosing the design matrix - there is always more than one choice which is correct provided it is interpreted correctly.

Design matrices for Affymetrix or single-color arrays can be created using the function model.matrix which is part of the R base package. The function modelMatrix is provided to assist with creation of an appropriate design matrix for two-color microarray experiments. For direct two-color designs, without a common reference, the design matrix often needs to be created by hand.

Making Comparisons of Interest

Once a linear model has been fit using an appropriate design matrix, the command makeContrasts may be used to form a contrast matrix to make comparisons of interest. The fit and the contrast matrix are used by contrasts.fit to compute fold changes and t-statistics for the contrasts of interest. This is a way to compute all possible pairwise comparisons between treatments for example in an experiment which compares many treatments to a common reference.

Assessing Differential Expression

After fitting a linear model, the standard errors are moderated using a simple empirical Bayes model using eBayes or treat. A moderated t-statistic and a log-odds of differential expression is computed for each contrast for each gene. treat tests whether log-fold-changes are greater than a threshold rather than merely different to zero.

eBayes and treat use internal functions squeezeVar, fitFDist, tmixture.matrix and tmixture.vector.

Summarizing Model Fits

After the above steps the results may be displayed or further processed using:

topTable

Presents a list of the genes most likely to be differentially expressed for a given contrast or set of contrasts.

topTableF

Presents a list of the genes most likely to be differentially expressed for a given set of contrasts. Equivalent to topTable with coef set to all the coefficients, coef=1:ncol(fit).

volcanoplot

Volcano plot of fold change versus the B-statistic for any fitted coefficient.

plotlines

Plots fitted coefficients or log-intensity values for time-course data.

genas

Estimates and plots biological correlation between two coefficients.

write.fit

Writes an MarrayLM object to a file. Note that if fit is an MArrayLM object, either write.fit or write.table can be used to write the results to a delimited text file.

For multiple testing functions which operate on linear model fits, see 08.Tests.

Model Selection

selectModel provides a means to choose between alternative linear models using AIC or BIC information criteria.

Author(s)

Gordon Smyth

References

Phipson, B, Lee, S, Majewski, IJ, Alexander, WS, and Smyth, GK (2016). Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Annals of Applied Statistics 10, 946-963. http://projecteuclid.org/euclid.aoas/1469199900

Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology, 3, No. 1, Article 3. https://gksmyth.github.io/pubs/ebayes.pdf

Smyth, G. K., Michaud, J., and Scott, H. (2005). The use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 21(9), 2067-2075.

See Also

01.Introduction, 02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

This page gives an overview of the LIMMA functions fit linear models to two-color microarray data in terms of the log-intensities rather than log-ratios.

The function intraspotCorrelation estimates the intra-spot correlation between the two channels. The regression function lmscFit takes the correlation as an argument and fits linear models to the two-color data in terms of the individual log-intensities. The output of lmscFit is an MArrayLM object just the same as from lmFit, so inference proceeds in the same way as for log-ratios once the linear model is fitted. See 06.LinearModels.

The function targetsA2C converts two-color format target data frames to single channel format, i.e, converts from array-per-line to channel-per-line, to facilitate the formulation of the design matrix.

Author(s)

Gordon Smyth

See Also

01.Introduction, 02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

LIMMA provides a number of functions for multiple testing across both contrasts and genes. The starting point is an MArrayLM object, called fit say, resulting from fitting a linear model and running eBayes and, optionally, contrasts.fit. See 06.LinearModels or 07.SingleChannel for details.

Multiple testing across genes and contrasts

The key function is decideTests. This function writes an object of class TestResults, which is basically a matrix of -1, 0 or 1 elements, of the same dimension as fit$coefficients, indicating whether each coefficient is significantly different from zero. A number of different multiple testing strategies are provided. decideTests calls classifyTestsF to implement the nested F-test strategt.

selectModel chooses between linear models for each probe using AIC or BIC criteria. This is an alternative to hypothesis testing and can choose between non-nested models.

A number of other functions are provided to display the results of decideTests. The functions heatDiagram (or the older version heatdiagram displays the results in a heat-map style display. This allows visual comparison of the results across many different conditions in the linear model.

The functions vennCounts and vennDiagram provide Venn diagram style summaries of the results.

Summary and show method exists for objects of class TestResults.

The results from decideTests can also be included when the results of a linear model fit are written to a file using write.fit.

Gene Set Tests

Competitive gene set testing for an individual gene set is provided by wilcoxGST or geneSetTest, which permute genes. The gene set can be displayed using barcodeplot.

Self-contained gene set testing for an individual set is provided by roast, which uses rotation technology, analogous to permuting arrays.

Gene set enrichment analysis for a large database of gene sets is provided by romer. topRomer is used to rank results from romer.

The functions alias2Symbol, alias2SymbolTable and alias2SymbolUsingNCBI are provided to help match gene sets with microarray probes by way of official gene symbols.

Global Tests

The function genas can test for associations between two contrasts in a linear model.

Given a set of p-values, the function propTrueNull can be used to estimate the proportion of true null hypotheses.

When evaluating test procedures with simulated or known results, the utility function auROC can be used to compute the area under the Receiver Operating Curve for the test results for a given probe.

Author(s)

Gordon Smyth

See Also

01.Introduction, 02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

This page gives an overview of the LIMMA functions available for microarray quality assessment and diagnostic plots.

This package provides an anova method which is designed for assessing the quality of an array series or of a normalization method. It is not designed to assess differential expression of individual genes. anova uses utility functions bwss and bwss.matrix.

The function arrayWeights estimates the empirical reliability of each array following a linear model fit.

Diagnostic plots can be produced by

imageplot

Produces a spatial picture of any spot-specific measure from an array image. If the log-ratios are plotted, then this produces an in-silico representation of the well known false-color TIFF image of an array. imageplot3by2 will write imageplots to files, six plots to a page.

plotFB

Plots foreground versus background log-intensies.

plotMD

Mean-difference plots. Very versatile plot. For two color arrays, this plots the M-values vs A-values. For single channel technologies, this plots one column of log-expression values vs the average of the other columns. For fitted model objects, this plots a log-fold-change versus average log-expression. mdplot can also be useful for comparing two one-channel microarrays.

plotMA

MA-plots, essentially the same as mean-difference plots. plotMA3by2 will write MA-plots to files, six plots to a page.

plotWithHighlights

Scatterplots with highlights. This is the underlying engine for plotMD and plotMA.

plotPrintTipLoess

Produces a grid of MA-plots, one for each print-tip group on an array, together with the corresponding lowess curve. Intended to help visualize print-tip loess normalization.

plotPrintorder

For an array, produces a scatter plot of log-ratios or log-intensities by print order.

plotDensities

Individual channel densities for one or more arrays. An essential plot to accompany between array normalization, especially quantile normalization.

plotMDS

Multidimensional scaling plot for a set of arrays. Useful for visualizing the relationship between the set of samples.

plotSA

Sigma vs A plot. After a linear model is fitted, this checks constancy of the variance with respect to intensity level.

plotPrintTipLoess uses utility functions gridr and gridc. plotDensities uses utility function RG.MA.

Author(s)

Gordon Smyth

See Also

01.Introduction, 02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

This page gives an overview of the LIMMA functions for gene set testing and pathway analysis.

roast

Self-contained gene set testing for one set. Uses zscoreT to normalize t-statistics.

mroast

Self-contained gene set testing for many sets. Uses zscoreT to normalize t-statistics.

fry

Fast approximation to mroast, especially useful when heteroscedasticity of genes can be ignored.

camera

Competitive gene set testing.

cameraPR

Competitive gene set testing with a pre-ranked gene set.

romer and topRomer

Gene set enrichment analysis.

ids2indices

Convert gene sets consisting of vectors of gene identifiers into a list of indices suitable for use in the above functions.

alias2Symbol, alias2SymbolTable and alias2SymbolUsingNCBI

Convert gene symbols or aliases to current official symbols.

geneSetTest or wilcoxGST

Simple gene set testing based on gene or probe permutation.

barcodeplot

Enrichment plot of a gene set.

goana and topGO

Gene ontology over-representation analysis of gene lists using Entrez Gene IDs. goana can work directly on a fitted model object or on one or more lists of genes.

kegga and topKEGG

KEGG pathway over-representation analysis of gene lists using Entrez Gene IDs. kegga can work directly on a fitted model object or on one or more lists of genes.

Author(s)

Gordon Smyth

See Also

01.Introduction, 02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

This page gives an overview of LIMMA functions to analyze RNA-seq data.

voom

Transform RNA-seq or ChIP-seq counts to log counts per million (log-cpm) with associated precision weights. After this tranformation, RNA-seq or ChIP-seq data can be analyzed using the same functions as would be used for microarray data.

voomWithQualityWeights

Combines the functionality of voom and arrayWeights.

diffSplice

Test for differential exon usage between experimental conditions.

topSplice

Show a data.frame of top results from diffSplice.

plotSplice

Plot results from diffSplice.

plotExons

Plot logFC for individual exons for a given gene.

References

Law, CW, Chen, Y, Shi, W, Smyth, GK (2014). Voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biology 15, R29. doi:10.1186/gb-2014-15-2-r29

Ritchie, ME, Phipson, B, Wu, D, Hu, Y, Law, CW, Shi, W, and Smyth, GK (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Research 43, e47. doi:10.1093/nar/gkv007

See Also

See also the edgeR package for normalization and data summaries of RNA-seq data, as well as for alternative differential expression methods based on the negative binomial distribution. voom accepts DGEList objects and normalization factors from edgeR.

The edgeR function voomLmFit is a drop-in replacement for either voom or voomWithQualityWeights.

01.Introduction, 02.Classes, 03.ReadingData, 04.Background, 05.Normalization, 06.LinearModels, 07.SingleChannel, 08.Tests, 09.Diagnostics, 10.GeneSetTests, 11.RNAseq

Description

Maps gene alias names to official gene symbols.

Usage

alias2Symbol(alias, species = "Hs", expand.symbols = FALSE)
alias2SymbolTable(alias, species = "Hs")
alias2SymbolUsingNCBI(alias, gene.info.file,
                      required.columns = c("GeneID","Symbol","description"))

Arguments

Details

Aliases are mapped via NCBI Entrez Gene identity numbers using Bioconductor organism packages.

alias2Symbol maps a set of aliases to a set of symbols, without necessarily preserving order. The output vector may be longer or shorter than the original vector, because some aliases might not be found and some aliases may map to more than one symbol.

alias2SymbolTable returns of vector of the same length as the vector of aliases. If an alias maps to more than one symbol, then the one with the lowest Entrez ID number is returned. If an alias can't be mapped, then NA is returned.

species can be any character string XX for which an organism package org.XX.eg.db exists and is installed. The only requirement of the organism package is that it contains objects org.XX.egALIAS2EG and org.XX.egSYMBOL linking the aliases and symbols to Entrez Gene Ids. At the time of writing, the following organism packages are available from Bioconductor 3.16:

alias2SymbolUsingNCBI is analogous to alias2SymbolTable but uses a gene-info file from NCBI instead of a Bioconductor organism package. It also gives the option of returning multiple columns from the gene-info file. NCBI gene-info files can be downloaded from https://ftp.ncbi.nlm.nih.gov/gene/DATA/GENE_INFO/. For example, the human file is https://ftp.ncbi.nlm.nih.gov/gene/DATA/GENE_INFO/Mammalia/Homo_sapiens.gene_info.gz and the mouse file is ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/GENE_INFO/Mammalia/Mus_musculus.gene_info.gz.

Value

alias2Symbol and alias2SymbolTable produce a character vector of gene symbols. alias2SymbolTable returns a vector of the same length and order as alias, including NA values where no gene symbol was found. alias2Symbol returns an unordered vector that may be longer or shorter than alias.

alias2SymbolUsingNCBI returns a data.frame with rows corresponding to the entries of alias and columns as specified by required.columns.

Author(s)

Gordon Smyth and Yifang Hu

See Also

This function is often used to assist gene set testing, see 10.GeneSetTests.

Examples

alias2Symbol(c("PUMA","NOXA","BIM"), species="Hs")
alias2Symbol("RS1", expand=TRUE)

Description

Analysis of variance method for objects of class MAList. Produces an ANOVA table useful for quality assessment by decomposing between and within gene sums of squares for a series of replicate arrays. This method produces a single ANOVA Table rather than one for each gene and is not used to identify differentially expressed genes.

Usage

anova(object,design=NULL,ndups=2,...)

Arguments

object

object of class MAList. Missing values in the M-values are not allowed.

design

numeric vector or single-column matrix containing the design matrix for linear model. The length of the vector or the number of rows of the matrix should agree with the number of columns of M.

ndups

number of duplicate spots. Each gene is printed ndups times in adjacent spots on each array.

...

other arguments are not used

Details

This function aids in quality assessment of microarray data and in the comparison of normalization methodologies. It applies only to replicated two-color experiments in which all the arrays are hybridized with the same RNA targets, possibly with dye-swaps, so the design matrix should have only one column. The function has not been heavily used and is somewhat experimental.

Value

An object of class anova containing rows for between genes, between arrays, gene x array interaction, and between duplicate with array sums of squares. Variance components are estimated for each source of variation.

Note

This function does not give valid results in the presence of missing M-values.

Author(s)

Gordon Smyth

See Also

MAList-class, bwss.matrix, anova.

An overview of quality assessment and diagnostic functions in LIMMA is given by 09.Diagnostics.

Description

Estimate relative quality weights for each array or group in a multi-array experiment.

Usage

arrayWeights(object, design = NULL, weights = NULL,
     var.design = NULL, var.group = NULL, prior.n = 10,
     method = "auto", maxiter = 50, tol = 1e-5, trace = FALSE)

Arguments

Details

The relative reliability of each array is estimated by measuring how well the expression values for that array follow the linear model. Arrays that tend to have larger residuals are assigned lower weights.

The basic method is described by Ritchie et al (2006) and the extension to custom variance models by Liu et al (2015). A weighted linear model is fitted to the expression values for each gene. The variance model is fitted to the squared residuals from the linear model fit and is updated either by full REML scoring iterations (method="reml") or using an efficient gene-by-gene update algorithm (method="genebygene"). The final estimates of these array variances are converted to weights. The gene-by-gene algorithm is described by Ritchie et al (2006) while the REML algorithm is an adaption of the algorithm of Smyth (2002).

For stability, the array weights are squeezed slightly towards equality. This is done by adding a prior likelihood corresponding to unit array weights equivalent to prior.n genes. The gene-by-gene algorithm is started from the prior genes while the REML algorithm adds the prior to the log-likelihood derivatives.

By default, arrayWeights chooses between the REML and gene-by-gene algorithms automatically (method="auto"). REML is chosen if there are no prior weights or missing values and otherwise the gene-by-gene algorithm is used.

The input object is interpreted as for lmFit and getEAWP. In particular, the arguments design and weights will be extracted from the data object if available and do not normally need to be set explicitly in the call; if any of these are set in the call then they will over-ride the slots or components in the data object.

Value

A numeric vector of array weights, which multiply to 1.

Author(s)

Matthew Ritchie and Gordon Smyth

References

Liu, R., Holik, A. Z., Su, S., Jansz, N., Chen, K., Leong, H. S., Blewitt, M. E., Asselin-Labat, M.-L., Smyth, G. K., Ritchie, M. E. (2015). Why weight? Combining voom with estimates of sample quality improves power in RNA-seq analyses. Nucleic Acids Research 43, e97. doi:10.1093/nar/gkv412

Ritchie, M. E., Diyagama, D., Neilson, van Laar, R., J., Dobrovic, A., Holloway, A., and Smyth, G. K. (2006). Empirical array quality weights in the analysis of microarray data. BMC Bioinformatics 7, 261. doi:10.1186/1471-2105-7-261

Smyth, G. K. (2002). An efficient algorithm for REML in heteroscedastic regression. Journal of Computational and Graphical Statistics 11, 836-847. https://gksmyth.github.io/pubs/remlalgo.pdf

See Also

arrayWeightsQuick, voomWithQualityWeights

An overview of linear model functions in limma is given by 06.LinearModels.

Examples

ngenes <- 1000
narrays <- 6
y <- matrix(rnorm(ngenes*narrays), ngenes, narrays)
var.group <- c(1,1,1,2,2,2)
y[,var.group==1] <- 2*y[,var.group==1]
arrayWeights(y, var.group=var.group)

Description

Estimates relative quality weights for each array in a multi-array experiment with replication.

Usage

arrayWeightsQuick(y, fit)

Arguments

Details

Estimates the relative reliability of each array by measuring how well the expression values for that array follow the linear model.

This is a quick and dirty version of arrayWeights.

Value

Numeric vector of weights of length ncol(fit).

Author(s)

Gordon Smyth

References

Ritchie, M. E., Diyagama, D., Neilson, van Laar, R., J., Dobrovic, A., Holloway, A., and Smyth, G. K. (2006). Empirical array quality weights in the analysis of microarray data. BMC Bioinformatics 7, 261. doi:10.1186/1471-2105-7-261

See Also

See arrayWeights. An overview of LIMMA functions for reading data is given in 03.ReadingData.

Examples

## Not run: 
fit <- lmFit(y, design)
arrayWeightsQuick(y, fit)

## End(Not run)

Description

Turn a MArrayLM object into a data.frame.

Usage

## S3 method for class 'MArrayLM'
as.data.frame(x, row.names = NULL, optional = FALSE, ...)

Arguments

Details

This method combines all the components of x which have a row for each probe on the array into a data.frame.

Value

A data.frame.

Author(s)

Gordon Smyth

See Also

as.data.frame in the base package.

02.Classes gives an overview of data classes used in LIMMA. 06.LinearModels gives an overview of linear model functions in LIMMA.

Description

Convert marrayNorm Object to an MAList Object

Usage

as.MAList(object)

Arguments

Details

The marrayNorm class is defined in the marray package. This function converts a normalized two color microarray data object created by the marray package into the corresponding limma data object.

Note that such conversion is not necessary to access the limma linear modelling functions, because lmFit will operate on a marrayNorm data object directly.

Value

Object of class MAList

Author(s)

Gordon Smyth

See Also

02.Classes gives an overview of all the classes defined by this package.

The marrayNorm class is defined in the marray package.

Description

Turn a microarray data object into a numeric matrix by extracting the expression values.

Usage

## S3 method for class 'MAList'
as.matrix(x,...)

Arguments

Details

These methods extract the matrix of log-ratios, for MAList or marrayNorm objects, or the matrix of expression values for other expression objects such as EList or ExressionSet. For MArrayLM objects, the matrix of fitted coefficients is extracted.

These methods involve loss of information, so the original data object is not recoverable.

Value

A numeric matrix.

Author(s)

Gordon Smyth

See Also

as.matrix in the base package or exprs in the Biobase package.

02.Classes gives an overview of data classes used in LIMMA.

Description

Convert probe-weights or array-weights to a matrix of weights.

Usage

asMatrixWeights(weights, dim)

Arguments

Details

This function converts a vector or probe-weights or a vector of array-weights to a matrix of the correct size. Probe-weights are repeated across rows while array-weights are repeated down the columns. If weights has length equal to the number of probes, it is assumed to contain probe-weights. If it has length equal to the number of arrays, it is assumed to contain array-weights. If the number of probes is equal to the number of arrays, then weights is assumed to contain array-weights if it is a row-vector of the correct size, i.e., if it is a matrix with one row.

This function is used internally by the linear model fitting functions in limma.

Value

Numeric matrix of dimension dim.

Author(s)

Gordon Smyth

See Also

modifyWeights.

An overview of functions in LIMMA used for fitting linear models is given in 06.LinearModels.

Examples

asMatrixWeights(1:3,c(4,3))
asMatrixWeights(1:4,c(4,3))

Description

Compute exact area under the ROC for empirical data.

Usage

auROC(truth, stat=NULL)

Arguments

Details

A receiver operating curve (ROC) is a plot of sensitivity (true positive rate) versus 1-specificity (false positive rate) for a statistical test or binary classifier. The area under the ROC is a well accepted measure of test performance. It is equivalent to the probability that a randomly chosen pair of cases is corrected ranked.

Here we consider a test statistic stat, with larger values being more significant, and a vector truth indicating whether the alternative hypothesis is in fact true. truth==TRUE or truth==1 indicates a true discovery and truth=FALSE or truth=0 indicates a false discovery. Correct ranking here means that truth[i] is greater than or equal to truth[j] when stat[i] is greater than stat[j]. The function computes the exact area under the empirical ROC curve defined by truth when ordered by stat.

If stat contains ties, then auROC returns the average area under the ROC for all possible orderings of truth for tied stat values.

The area under the curve is undefined if truth is all TRUE or all FALSE or if truth or stat contain missing values.

Value

Numeric value between 0 and 1 giving area under the curve, 1 being perfect and 0 being the minimum.

Author(s)

Gordon Smyth

Examples

auROC(c(1,1,0,0,0))
truth <- rbinom(30,size=1,prob=0.2)
stat <- rchisq(30,df=2)
auROC(truth,stat)

Description

Condense a microarray data object so that technical replicate arrays are replaced with (weighted) averages.

Usage

## Default S3 method:
avearrays(x, ID=colnames(x), weights=NULL)
## S3 method for class 'MAList'
avearrays(x, ID=colnames(x), weights=x$weights)
## S3 method for class 'EList'
avearrays(x, ID=colnames(x), weights=x$weights)

Arguments

Details

A new data object is computed in which technical replicate arrays are replaced by their (weighted) averages.

For an MAList object, the components M and A are both averaged in this way, as are weights and any matrices found in object$other.

EList objects are similar, except that the E component is averaged instead of M and A.

If x is of mode "character", then the replicate values are assumed to be equal and the first is taken as the average.

Value

A data object of the same class as x with a column for each unique value of ID.

Author(s)

Gordon Smyth

See Also

avereps.

02.Classes gives an overview of data classes used in LIMMA.

Examples

x <- matrix(rnorm(8*3),8,3)
colnames(x) <- c("a","a","b")
avearrays(x)

Description

Condense a microarray data object so that values for within-array replicate spots are replaced with their average.

Usage

## Default S3 method:
avedups(x, ndups=2, spacing=1, weights=NULL)
## S3 method for class 'MAList'
avedups(x, ndups=x$printer$ndups, spacing=x$printer$spacing, weights=x$weights)
## S3 method for class 'EList'
avedups(x, ndups=x$printer$ndups, spacing=x$printer$spacing, weights=x$weights)

Arguments

Details

A new data object is computed in which each probe is represented by the (weighted) average of its duplicate spots. For an MAList object, the components M and A are both averaged in this way. For an EList object, the component E is averaged in this way.

If x is of mode "character", then the duplicate values are assumed to be equal and the first is taken as the average.

Value

A data object of the same class as x with 1/ndups as many rows.

Author(s)

Gordon Smyth

See Also

avereps.

02.Classes gives an overview of data classes used in LIMMA.

Description

Condense a microarray data object so that values for within-array replicate probes are replaced with their average.

Usage

## Default S3 method:
avereps(x, ID=rownames(x), ...)
## S3 method for class 'MAList'
avereps(x, ID=NULL, ...)
## S3 method for class 'EList'
avereps(x, ID=NULL, ...)

Arguments

Details

A new data object is computed in which each probe ID is represented by the average of its replicate spots or features.

For an MAList object, the components M and A are both averaged in this way, as are weights and any matrices found in object$other. For an MAList object, ID defaults to MA$genes$ID is that exists, otherwise to rownames(MA$M).

EList objects are similar, except that the E component is averaged instead of M and A.

If x is of mode "character", then the replicate values are assumed to be equal and the first is taken as the average.

Value

A data object of the same class as x with a row for each unique value of ID.

Note

This function should only be applied to normalized log-expression values, and not to raw unlogged expression values. It will generate an error message if applied to RGList or EListRaw objects.

Author(s)

Gordon Smyth

See Also

avedups, avearrays. Also rowsum in the base package.

02.Classes gives an overview of data classes used in LIMMA.

Examples

x <- matrix(rnorm(8*3),8,3)
colnames(x) <- c("S1","S2","S3")
rownames(x) <- c("b","a","a","c","c","b","b","b")
avereps(x)

Description

Background correct microarray expression intensities.

Usage

backgroundCorrect(RG, method="auto", offset=0, printer=RG$printer,
                  normexp.method="saddle", verbose=TRUE)
backgroundCorrect.matrix(E, Eb=NULL, method="auto", offset=0, printer=NULL,
                  normexp.method="saddle", verbose=TRUE)

Arguments

Details

This function implements the background correction methods reviewed or developed in Ritchie et al (2007) and Silver at al (2009). Ritchie et al (2007) recommend method="normexp" whenever RG contains local background estimates. Silver et al (2009) shows that either normexp.method="mle" or normexp.method="saddle" are excellent options for normexp. If RG contains morphological background estimates instead (available from SPOT or GenePix image analysis software), then method="subtract" performs well.

If method="none" then no correction is done, i.e., the background intensities are treated as zero. If method="subtract" then the background intensities are subtracted from the foreground intensities. This is the traditional background correction method, but is not necessarily recommended. If method="movingmin" then the background estimates are replaced with the minimums of the backgrounds of the spot and its eight neighbors, i.e., the background is replaced by a moving minimum of 3x3 grids of spots.

The remaining methods are all designed to produce positive corrected intensities. If method="half" then any intensity which is less than 0.5 after background subtraction is reset to be equal to 0.5. If method="minimum" then any intensity which is zero or negative after background subtraction is set equal to half the minimum of the positive corrected intensities for that array. If method="edwards" a log-linear interpolation method is used to adjust lower intensities as in Edwards (2003). If method="normexp" a convolution of normal and exponential distributions is fitted to the foreground intensities using the background intensities as a covariate, and the expected signal given the observed foreground becomes the corrected intensity. This results in a smooth monotonic transformation of the background subtracted intensities such that all the corrected intensities are positive.

The normexp method is available in a number of variants depending on how the model parameters are estimated, and these are selected by normexp.method. Here "saddle" gives the saddle-point approximation to maximum likelihood from Ritchie et al (2007) and improved by Silver et al (2009), "mle" gives exact maximum likelihood from Silver at al (2009), "rma" gives the background correction algorithm from the RMA-algorithm for Affymetrix microarray data as implemented in the affy package, and "rma75" gives the RMA-75 method from McGee and Chen (2006). In practice "mle" performs well and is nearly as fast as "saddle", but "saddle" is the default for backward compatibility. See normexp.fit for more details.

The offset can be used to add a constant to the intensities before log-transforming, so that the log-ratios are shrunk towards zero at the lower intensities. This may eliminate or reverse the usual 'fanning' of log-ratios at low intensities associated with local background subtraction.

Background correction (background subtraction) is also performed by the normalizeWithinArrays method for RGList objects, so it is not necessary to call backgroundCorrect directly unless one wants to use a method other than simple subtraction. Calling backgroundCorrect before normalizeWithinArrays will over-ride the default background correction.

Value

A matrix, EListRaw or RGList object in which foreground intensities have been background corrected and any components containing background intensities have been removed.

Author(s)

Gordon Smyth

References

Edwards, D. E. (2003). Non-linear normalization and background correction in one-channel cDNA microarray studies Bioinformatics 19, 825-833.

McGee, M., and Chen, Z. (2006). Parameter estimation for the exponential-normal convolution model for background correction of Affymetrix GeneChip data. Stat Appl Genet Mol Biol, Volume 5, Article 24.

Ritchie, M. E., Silver, J., Oshlack, A., Silver, J., Holmes, M., Diyagama, D., Holloway, A., and Smyth, G. K. (2007). A comparison of background correction methods for two-colour microarrays. Bioinformatics 23, 2700-2707. http://bioinformatics.oxfordjournals.org/content/23/20/2700

Silver, J., Ritchie, M. E., and Smyth, G. K. (2009). Microarray background correction: maximum likelihood estimation for the normal-exponential convolution model. Biostatistics 10, 352-363. http://biostatistics.oxfordjournals.org/content/10/2/352

See Also

kooperberg, neqc.

An overview of background correction functions is given in 04.Background.

Examples

RG <- new("RGList", list(R=c(1,2,3,4),G=c(1,2,3,4),Rb=c(2,2,2,2),Gb=c(2,2,2,2)))
backgroundCorrect(RG)
backgroundCorrect(RG, method="half")
backgroundCorrect(RG, method="minimum")
backgroundCorrect(RG, offset=5)

Description

Display the enrichment of one or two gene sets in a ranked gene list.

Usage

barcodeplot(statistics, index = NULL, index2 = NULL, gene.weights = NULL,
            weights.label = "Weight", labels = c("Down","Up"),
            quantiles = c(-1,1)*sqrt(2), col.bars = NULL, alpha = 0.4,
            worm = TRUE, span.worm = 0.45, xlab = "Statistic", ...)

Arguments

Details

The function displays the enrichment of a specified gene set signature in a ranked list of genes. The vector statistics defines the ranking of the population of genes. This vector can represent any useful ranking but often it provides t-statistics or a log-fold-changes arising from a differential analysis. The gene set signature is defined either by index and index2 or by gene.weights.

The signature can be either unidirectional or bidirectional. A unidirectional signature is a simple set of genes (defined by index), optionally accompanied by a set of positive magnitude scores (specified by gene.weights). Typically this is a set of genes representing a pathway or biological process that are expected to be co-regulated in the same direction. A bidirectional signature consists of a set of up-genes and a set of down-genes (specified by index and index2 respectively) or, more generally, a set of genes with accompanying magnitude scores that are either positive or negative (specified by gene.weights).

Technically, this function plots the positions of one or two gene sets in a ranked list of statistics. If there are two sets, then one is considered to be the positive set and the other the down set. For example, the first set and second sets often correspond to genes that are expected to be up- or down-regulated respectively. The function can optionally display varying weights for different genes, for example log-fold-changes from a previous experiment.

The statistics are ranked left to right from smallest to largest. The ranked statistics are represented by a shaded bar or bed, and the positions of the specified subsets are marked by vertical bars, forming a pattern like a barcode. An enrichment worm optionally shows the relative enrichment of the vertical bars in each part of the plot. The worm is computed by the tricubeMovingAverage function.

Barcode plots are often used in conjunction with gene set tests, and show the enrichment of gene sets amongst high or low ranked genes. They were inspired by the set location plot of Subramanian et al (2005), with a number of enhancements, especially the ability to plot positive and negative sets simultaneously. Barcode plots first appeared in the literature in Lim et al (2009). More recent examples can be seen in Liu et al (2014), Sheikh et al (2015), Witkowski et al (2015) and Ng et al (2015).

The function can be used with any of four different calling sequences:

• index is specified, but not index2 or gene.weights. Single direction plot.

• index and index2 are specified. Two directional plot.

• index and gene.weights are specified. gene.weights must have same length as statistics[index]. Plot will be two-directional if gene.weights contains positive and negative values.

• gene.weights is specified by not index or index2. gene.weights must have same length as statistics. Plot will be two-directional if gene.weights contains positive and negative values.

Value

No value is returned but a plot is produced as a side effect.

Author(s)

Yifang Hu, Gordon Smyth and Di Wu

References

Ng, AP, Hu, Y, Metcalf, D, Hyland, CD, Ierino, H, Phipson, B, Wu, D, Baldwin, TM, Kauppi, M, Kiu, H, Di, Rago, L, Hilton, DJ, Smyth, GK, Alexander, WS (2015). Early lineage priming by trisomy of Erg leads to myeloproliferation in a down syndrome model. PLOS Genetics 11, e1005211. doi:10.1371/journal.pgen.1005211

Lim E, Vaillant F, Wu D, Forrest NC, Pal B, Hart AH, Asselin-Labat ML, Gyorki DE, Ward T, Partanen A, Feleppa F, Huschtscha LI, Thorne HJ; kConFab; Fox SB, Yan M, French JD, Brown MA, Smyth GK, Visvader JE, and Lindeman GJ (2009). Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nature Medicine 15, 907-913. doi:10.1038/nm.2000

Liu, GJ, Cimmino, L, Jude, JG, Hu, Y, Witkowski, MT, McKenzie, MD, Kartal-Kaess, M, Best, SA, Tuohey, L, Liao, Y, Shi, W, Mullighan, CG, Farrar, MA, Nutt, SL, Smyth, GK, Zuber, J, and Dickins, RA (2014). Pax5 loss imposes a reversible differentiation block in B progenitor acute lymphoblastic leukemia. Genes & Development 28, 1337-1350. doi:10.1101/gad.240416.114

Sheikh, B, Lee, S, El-saafin, F, Vanyai, H, Hu, Y, Pang, SHM, Grabow, S, Strasser, A, Nutt, SL, Alexander, WS, Smyth, GK, Voss, AK, and Thomas, T (2015). MOZ regulates B cell progenitors in mice, consequently, Moz haploinsufficiency dramatically retards MYC-induced lymphoma development. Blood 125, 1910-1921. doi:10.1182/blood-2014-08-594655

Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, and Mesirov JP (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102, 15545-15550.

Witkowski, MT, Cimmino, L, Hu, Y, Trimarchi, T, Tagoh, H, McKenzie, MD, Best, SA, Tuohey, L, Willson, TA, Nutt, SL, Meinrad Busslinger, M, Aifantis, I, Smyth, GK, and Dickins, RA (2015). Activated Notch counteracts Ikaros tumor suppression in mouse and human T cell acute lymphoblastic leukemia. Leukemia 29, 1301-1311. doi:10.1038/leu.2015.27

See Also

tricubeMovingAverage, roast, camera, romer, geneSetTest

There is a topic page on 10.GeneSetTests.

Examples

stat <- rnorm(100)
sel <- 1:10
sel2 <- 11:20
stat[sel] <- stat[sel]+1
stat[sel2] <- stat[sel2]-1

# One directional
barcodeplot(stat, index = sel)

# Two directional
barcodeplot(stat, index = sel, index2 = sel2)

# Second set can be indicated by negative weights
barcodeplot(stat, index = c(sel,sel2), gene.weights = c(rep(1,10), rep(-1,10)))

# Two directional with unequal weights
w <- rep(0,100)
w[sel] <- runif(10)
w[sel2] <- -runif(10)
barcodeplot(stat, gene.weights = w, weights.label = "logFC")

# One directional with unequal weights
w <- rep(0,100)
w[sel2] <- -runif(10)
barcodeplot(stat, gene.weights = w, weights.label = "logFC", col.bars = "dodgerblue")

Description

Estimates weights which account for biological variation and technical variation resulting from varying bead numbers.

Usage

beadCountWeights(y, x, design = NULL, bead.stdev = NULL, bead.stderr = NULL,
                 nbeads = NULL, array.cv = TRUE, scale = FALSE)

Arguments

Details

This function estimates optimum weights using the bead statistics for each probe for an Illumina expression BeadChip. It can be used with any Illumina expression BeadChip, but is most likely to be useful with HumanHT-12 BeadChips.

Arguments x and y are both required. x contains the raw expression values and y contains the corresponding log2 values for the same probes and the same arrays after background correction and normalization. x and y be any type of object that can be coerced to a matrix, with rows corresponding to probes and columns to arrays. x and y must contain the same rows and columns in the same order.

The reliability of the normalized expression value for each probe on each array is measured by estimating its technical and biological variability. The bead number weights are the inverse sum of the technical and biological variances.

The technical variance for each probe on each array is inversely proportional to the number of beads and is estimated using array-specific bead-level coefficients of variation.

Coefficients of variation are calculated using raw expression values.

The biological variance for each probe across the arrays are estimated using a Newton iteration, with the assumption that the total residual deviance for each probe from lmFit is inversely proportional to the sum of the technical variance and biological variance.

Only one of bead.stdev or bead.stderr needs to be set. If bead.stdev is not provided, then it will be computed as bead.stderr * sqrt(nbeads).

If arguments bead.stdev and nbeads are not set explicitly in the call, then they will be extracted fromy$other$BEAD_STDEV and y$other$Avg_NBEADS. An EList object containing these components can be created by read.idat or read.ilmn, see the example code below.

Value

A list object with the following components:

Author(s)

Charity Law and Gordon Smyth

References

Law CW (2013). Precision weights for gene expression analysis. PhD Thesis. University of Melbourne, Australia. http://hdl.handle.net/11343/38150

See Also

read.ilmn, read.idat, neqc.

An overview of linear model functions in limma is given by 06.LinearModels.

Examples

## Not run: 
z <- read.ilmn(files="probesummaryprofile.txt",
     ctrfiles="controlprobesummary.txt",
     other.columns=c("BEAD_STDEV","Avg_NBEADS"))
y <- neqc(z)
x <- z[z$genes$Status=="regular",]
bcw <- beadCountWeights(y,x,design)
fit <- lmFit(y,design,weights=bcw$weights)
fit <- eBayes(fit)

## End(Not run)

Description

Form a block diagonal matrix from the given blocks.

Usage

blockDiag(...)

Arguments

Details

This function is sometimes useful for constructing a design matrix for a disconnected two-color microarray experiment in conjunction with modelMatrix.

Value

A block diagonal matrix with dimensions equal to the sum of the input dimensions

Author(s)

Gordon Smyth

See Also

modelMatrix

Examples

a <- matrix(1,3,2)
b <- matrix(2,2,2)
blockDiag(a,b)

Description

Sums of squares between and within groups. Allows for missing values.

Usage

bwss(x,group)

Arguments

Details

This is equivalent to one-way analysis of variance.

Value

A list with components

Author(s)

Gordon Smyth

See Also

bwss.matrix

Description

Sums of squares between and within the columns of a matrix. Allows for missing values. This function is called by the anova method for MAList objects.

Usage

bwss.matrix(x)

Arguments

Details

This is equivalent to a one-way analysis of variance where the columns of the matrix are the groups. If x is a matrix then bwss.matrix(x) is the same as bwss(x,col(x)) except for speed of execution.

Value

A list with components

Author(s)

Gordon Smyth

See Also

bwss, anova.MAList

Description

Test whether a set of genes is highly ranked relative to other genes in terms of differential expression, accounting for inter-gene correlation.

Usage

## Default S3 method:
camera(y, index, design, contrast = ncol(design), weights = NULL,
       use.ranks = FALSE, allow.neg.cor=FALSE, inter.gene.cor=0.01, trend.var = FALSE,
       sort = TRUE, ...)
## Default S3 method:
cameraPR(statistic, index, use.ranks = FALSE, inter.gene.cor=0.01, sort = TRUE, ...)
interGeneCorrelation(y, design)

Arguments

Details

camera and interGeneCorrelation implement methods proposed by Wu and Smyth (2012). camera performs a competitive test in the sense defined by Goeman and Buhlmann (2007). It tests whether the genes in the set are highly ranked in terms of differential expression relative to genes not in the set. It has similar aims to geneSetTest but accounts for inter-gene correlation. See roast for an analogous self-contained gene set test.

The function can be used for any microarray experiment which can be represented by a linear model. The design matrix for the experiment is specified as for the lmFit function, and the contrast of interest is specified as for the contrasts.fit function. This allows users to focus on differential expression for any coefficient or contrast in a linear model by giving the vector of test statistic values.

camera estimates p-values after adjusting the variance of test statistics by an estimated variance inflation factor. The inflation factor depends on estimated genewise correlation and the number of genes in the gene set.

By default, camera uses interGeneCorrelation to estimate the mean pair-wise correlation within each set of genes. camera can alternatively be used with a preset correlation specified by inter.gene.cor that is shared by all sets. This usually works best with a small value, say inter.gene.cor=0.01.

If inter.gene.cor=NA, then camera will estimate the inter-gene correlation for each set. In this mode, camera gives rigorous error rate control for all sample sizes and all gene sets. However, in this mode, highly co-regulated gene sets that are biological interpretable may not always be ranked at the top of the list.

With the default value inter.gene.cor=0.01, camera will rank biologically interpretable sets more highly. This gives a useful compromise between strict error rate control and interpretable gene set rankings.

cameraPR is a "pre-ranked" version of camera where the genes are pre-ranked according to a pre-computed statistic.

Value

camera and cameraPR return a data.frame with a row for each set and the following columns:

interGeneCorrelation returns a list with components:

Note

The default settings for inter.gene.cor and allow.neg.cor were changed to the current values in limma 3.29.6. Previously, the default was to estimate an inter-gene correlation for each set. To reproduce the earlier default, use allow.neg.cor=TRUE and inter.gene.cor=NA.

Author(s)

Di Wu and Gordon Smyth

References

Wu D, Smyth GK (2012). Camera: a competitive gene set test accounting for inter-gene correlation. Nucleic Acids Research 40, e133. doi:10.1093/nar/gks461

Goeman JJ, Buhlmann P (2007). Analyzing gene expression data in terms of gene sets: methodological issues. Bioinformatics 23, 980-987.

See Also

getEAWP

rankSumTestWithCorrelation, geneSetTest, roast, fry, romer, ids2indices.

There is a topic page on 10.GeneSetTests.

Examples

y <- matrix(rnorm(1000*6),1000,6)
design <- cbind(Intercept=1,Group=c(0,0,0,1,1,1))

# First set of 20 genes are genuinely differentially expressed
index1 <- 1:20
y[index1,4:6] <- y[index1,4:6]+1

# Second set of 20 genes are not DE
index2 <- 21:40
 
camera(y, index1, design)
camera(y, index2, design)

camera(y, list(set1=index1,set2=index2), design, inter.gene.cor=NA)
camera(y, list(set1=index1,set2=index2), design, inter.gene.cor=0.01)

# Pre-ranked version
fit <- eBayes(lmFit(y, design))
cameraPR(fit$t[,2], list(set1=index1,set2=index2))

Description

Combine a set of RGList, MAList, EList or EListRaw objects.

Usage

## S3 method for class 'RGList'
cbind(..., deparse.level=1)
## S3 method for class 'RGList'
rbind(..., deparse.level=1)

Arguments

Details

cbind combines data objects assuming the same probes in the same order but different arrays. rbind combines data objects assuming equivalent arrays, i.e., the same RNA targets, but different probes.

For cbind, the matrices of expression data from the individual objects are cbinded. The data.frames of target information, if they exist, are rbinded. The combined data object will preserve any additional components or attributes found in the first object to be combined. For rbind, the matrices of expression data are rbinded while the target information, in any, is unchanged.

Value

An RGList, MAList, EList or EListRaw object holding data from all the arrays and all genes from the individual objects.

Author(s)

Gordon Smyth

See Also

cbind in the base package.

03.ReadingData gives an overview of data input and manipulation functions in LIMMA.

Examples

M <- A <- matrix(11:14,4,2)
rownames(M) <- rownames(A) <- c("a","b","c","d")
colnames(M) <- colnames(A) <- c("A1","A2")
MA1 <- new("MAList",list(M=M,A=A))

M <- A <- matrix(21:24,4,2)
rownames(M) <- rownames(A) <- c("a","b","c","d")
colnames(M) <- colnames(A) <- c("B1","B2")
MA2 <- new("MAList",list(M=M,A=A))

cbind(MA1,MA2)

Description

Show the most recent changes from a package change log or NEWS file.

Usage

changeLog(n = 30, package = "limma")

Arguments

Details

The function will look for a file changelog.txt or ChangeLog in the top-level or doc directories of the installed package. Failing that, it will look for NEWS or NEWS.md in the top-level directory.

Note that changeLog does not write the content of NEWS.Rd, which is a structured file. Use news(package="limma") for that instead.

Value

No value is produced, but a number of lines of text are written to standard output.

Author(s)

Gordon Smyth

See Also

01.Introduction, news.

Examples

changeLog()
changeLog(package="statmod")

Description

Choose an optimal span, depending on the number of points, for lowess smoothing of variance trends.

Usage

chooseLowessSpan(n=1000, small.n=50, min.span=0.3, power=1/3)

Arguments

Details

The span is the proportion of points used for each of the local regressions. When there a few points, a large span should be used to ensure a smooth curve. When there are a large number of points, smaller spans can be used because each span window still contains good coverage. By default, the chosen span decreases as the cube-root of the number of points, a rule that is motivated by analogous rules to choose the number of bins for a histogram (Scott, 1979; Freedman & Diaconis, 1981; Hyndman, 1995).

The span returned is min.span + (1-min.span) * (small.n/n)^power except that the span is set to 1 for any n less than small.n. Note that the fitted lowess curve will still estimate a trend (i.e., will not be constant) even if span=1.

The function is tuned for smoothing of mean-variance trends, for which the trend is usually monotonic, so preference is given to moderately large spans. Even for the very large datasets, the span is always greater than min.span.

This function is used to create adaptive spans for voom, vooma and voomaLmFit where n is the number of genes in the analysis.

Value

A numeric vector of length 1 containing the span value.

Author(s)

Gordon Smyth

References

Freedman, D. and Diaconis, P. (1981). On the histogram as a density estimator: L_2 theory. Zeitschrift fur Wahrscheinlichkeitstheorie und verwandte Gebiete 57, 453-476.

Hyndman, R. J. (1995). The problem with Sturges' rule for constructing histograms. https://robjhyndman.com/papers/sturges.pdf.

Scott, D. W. (1979). On optimal and data-based histograms. Biometrika 66, 605-610.

See Also

loessFit, weightedLowess, lowess, loess.

vooma, eBayes, squeezeVar, fitFDistRobustly.

Examples

chooseLowessSpan(100)
chooseLowessSpan(1e6)

n <- 10:5000
span <- chooseLowessSpan(n)
plot(n,span,type="l",log="x")

Description

For each gene, classify a series of related t-statistics as significantly up or down using nested F-tests.

Usage

classifyTestsF(object, cor.matrix = NULL, df = Inf, p.value = 0.01, fstat.only = FALSE)

Arguments

Details

classifyTestsF implements the "nestedF" multiple testing option offered by decideTests. Users should generally use decideTests rather than calling classifyTestsF directly because, by itself, classifyTestsF does not incorporate any multiple testing adjustment across genes. Instead it simply tests across contrasts for each gene individually.

classifyTestsF uses a nested F-test approach giving particular attention to correctly classifying genes that have two or more significant t-statistics, i.e., which are differentially expressed in two or more conditions. For each row of tstat, the overall F-statistics is constructed from the t-statistics as for FStat. At least one constrast will be classified as significant if and only if the overall F-statistic is significant. If the overall F-statistic is significant, then the function makes a best choice as to which t-statistics contributed to this result. The methodology is based on the principle that any t-statistic should be called significant if the F-test is still significant for that row when all the larger t-statistics are set to the same absolute size as the t-statistic in question.

Compared to conventional multiple testing methods, the nested F-test approach achieves better consistency between related contrasts. (For example, if B is judged to be different from C, then at least one of B or C should be different to A.) The approach was first used by Michaud et al (2008). The nested F-test approach provides weak control of the family-wise error rate, i.e., it correctly controls the type I error rate of calling any contrast as significant if all the null hypotheses are true. In other words, it provides error rate control at the overall F-test level but does not provide strict error rate control at the individual contrast level.

Usually object is a limma linear model fitted object, from which a matrix of t-statistics can be extracted, but it can also be a numeric matrix of t-statistics. In either case, rows correspond to genes and columns to coefficients or contrasts. If object is a matrix, then it may be necessary to supply values for cor.matrix and df. The cor.matrix is the same as the correlation matrix of the coefficients from which the t-statistics were calculated and df is the degrees of freedom of the t-statistics. All statistics for the same gene must have the same degrees of freedom.

If fstat.only=TRUE, the classifyTestsF just returns the vector of overall F-statistics for each gene.

Value

If fstat.only=FALSE, then an object of class TestResults is returned. This is essentially a numeric matrix with elements -1, 0 or 1 depending on whether each t-statistic is classified as significantly negative, not significant or significantly positive respectively.

If fstat.only=TRUE, then a numeric vector of F-statistics is returned with attributes df1 and df2 giving the corresponding degrees of freedom.

Author(s)

Gordon Smyth

References

Michaud, J, Simpson, KM, Escher, R, Buchet-Poyau, K, Beissbarth, T, Carmichael, C, Ritchie, ME, Schutz, F, Cannon, P, Liu, M, Shen, X, Ito, Y, Raskind, WH, Horwitz, MS, Osato, M, Turner, DR, Speed, TP, Kavallaris, M, Smyth, GK, and Scott, HS (2008). Integrative analysis of RUNX1 downstream pathways and target genes. BMC Genomics 9, 363.

See Also

An overview of multiple testing functions is given in 08.Tests.

Examples

TStat <- matrix(c(0,10,0, 0,5,0, -4,-4,4, 2,2,2), 4, 3, byrow=TRUE)
colnames(TStat) <- paste0("Contrast",1:3)
rownames(TStat) <- paste0("Gene",1:4)
classifyTestsF(TStat, df=20)
FStat <- classifyTestsF(TStat, df=20, fstat.only=TRUE)
P <- pf(FStat, df1=attr(FStat,"df1"), df2=attr(FStat,"df2"), lower.tail=FALSE)
data.frame(F.Statistic=FStat,P.Value=P)

Description

Reform a design matrix so that one or more coefficients from the new matrix correspond to specified contrasts of coefficients from the old matrix.

Usage

contrastAsCoef(design, contrast=NULL, first=TRUE)

Arguments

Details

If the contrasts contained in the columns of contrast are not linearly dependent, then superfluous columns are dropped until the remaining matrix has full column rank. The number of retained contrasts is stored in qr$rank and the retained columns are given by qr$pivot.

Value

A list with components

Author(s)

Gordon Smyth

See Also

model.matrix in the stats package.

An overview of linear model functions in limma is given by 06.LinearModels.

Examples

design <- cbind(1,c(0,0,1,1,0,0),c(0,0,0,0,1,1))
cont <- c(0,-1,1)
design2 <- contrastAsCoef(design, cont)$design

#  Original coef[3]-coef[2] becomes coef[1]
y <- rnorm(6)
fit1 <- lm(y~0+design)
fit2 <- lm(y~0+design2)
coef(fit1)
coef(fit1) 
coef(fit2)

Description

Given a linear model fit to microarray data, compute estimated coefficients and standard errors for a given set of contrasts.

Usage

contrasts.fit(fit, contrasts=NULL, coefficients=NULL)

Arguments

Details

This function accepts input from any of the functions lmFit, lm.series, mrlm, gls.series or lmscFit. The function re-orientates the fitted model object from the coefficients of the original design matrix to any set of contrasts of the original coefficients. The coefficients, unscaled standard deviations and correlation matrix are re-calculated in terms of the contrasts.

The idea of this function is to fit a full-rank model using lmFit or equivalent, then use contrasts.fit to obtain coefficients and standard errors for any number of contrasts of the coefficients of the original model. Unlike the design matrix input to lmFit, which normally has one column for each treatment in the experiment, the matrix contrasts may have any number of columns and these are not required to be linearly independent. Methods of assessing differential expression, such as eBayes or classifyTestsF, can then be applied to fitted model object.

The coefficients argument provides a simpler way to specify the contrasts matrix when the desired contrasts are just a subset of the original coefficients.

Value

An list object of the same class as fit, usually MArrayLM. This is a list with components

Most other components found in fit are passed through unchanged, but t, p.value, lods, F and F.p.value will all be removed.

Note

For efficiency reasons, this function does not re-factorize the design matrix for each probe. A consequence is that, if the design matrix is non-orthogonal and the original fit included precision weights or missing values, then the unscaled standard deviations produced by this function are approximate rather than exact. The approximation is usually acceptable. If not, then the issue can be avoided by redefining the design matrix to fit the contrasts directly.

Even with precision weights, the results from contrasts.fit are always exact if the coefficients being compared are statistically independent. This will be true, for example, if the original fit was a oneway model without blocking and the group-means (no-intercept) parametrization was used for the design matrix.

Author(s)

Gordon Smyth

See Also

An overview of linear model functions in limma is given by 06.LinearModels.

Examples

#  Simulate gene expression data: 6 microarrays and 100 genes
#  with one gene differentially expressed in first 3 arrays
M <- matrix(rnorm(100*6,sd=0.3),100,6)
M[1,1:3] <- M[1,1:3] + 2
#  Design matrix corresponds to oneway layout, columns are orthogonal
design <- cbind(First3Arrays=c(1,1,1,0,0,0),Last3Arrays=c(0,0,0,1,1,1))
fit <- lmFit(M,design=design)
#  Would like to consider original two estimates plus difference between first 3 and last 3 arrays
contrast.matrix <- cbind(First3=c(1,0),Last3=c(0,1),"Last3-First3"=c(-1,1))
fit2 <- contrasts.fit(fit,contrast.matrix)
fit2 <- eBayes(fit2)
#  Large values of eb$t indicate differential expression
results <- decideTests(fit2, method="nestedF")
vennCounts(results)

Description

Determine the type (or status) of each spot in the gene list.

Usage

controlStatus(types, genes, spottypecol="SpotType", regexpcol, verbose=TRUE)

Arguments

Details

This function constructs a vector of status codes by searching for patterns in the gene list. The data frame genes contains gene IDs and should have as many rows as there are spots on the microarrays. Such a data frame is often read using readGAL. The data frame types has as many rows as you want to distinguish types of spots in the gene list. This data frame should contain a column or columns, the regexpcol columns, which have the same names as columns in genes and which contain patterns to match in the gene list. Another column, the spottypecol, contains the names of the spot types. Any other columns are assumed to contain plotting parameters, such as colors or symbols, to be associated with the spot types.

The patterns in the regexpcol columns are simplified regular expressions. For example, AA* means any string starting with AA, *AA means any code ending with AA, AA means exactly these two letters, *AA* means any string containing AA, AA. means AA followed by exactly one other character and AA\. means exactly AA followed by a period and no other characters. Any other regular expressions are allowed but the codes ^ for beginning of string and $ for end of string should not be included.

Note that the patterns are matched sequentially from first to last, so more general patterns should be included first. For example, it is often a good idea to include a default spot-type as the first line in types with pattern * for all regexpcol columns and default plotting parameters.

Value

Character vector specifying the type (or status) of each spot on the array. Attributes contain plotting parameters associated with each spot type.

Author(s)

Gordon Smyth

See Also

An overview of LIMMA functions for reading data is given in 03.ReadingData.

Examples

genes <- data.frame(
      ID=c("Control","Control","Control","Control","AA1","AA2","AA3","AA4"),
      Name=c("Ratio 1","Ratio 2","House keeping 1","House keeping 2",
             "Gene 1","Gene 2","Gene 3","Gene 4"))
types <- data.frame(
      SpotType=c("Gene","Ratio","Housekeeping"),
      ID=c("*","Control","Control"),
      Name=c("*","Ratio*","House keeping*"),
      col=c("black","red","blue"))
status <- controlStatus(types,genes)

Description

Create a heatmap of a matrix of log-expression values.

Usage

coolmap(x, cluster.by="de pattern", col=NULL,
        linkage.row="complete", linkage.col="complete", show.dendrogram="both", ...)

Arguments

Details

This function calls the heatmap.2 function in the gplots package with sensible argument settings for genomic log-expression data. The default settings for heatmap.2 are often not ideal for expression data, and overriding the defaults requires explicit calls to hclust and as.dendrogram as well as prior standardization of the data values. The coolmap function implements our preferred defaults for the two most common types of heatmaps. When clustering by relative expression (cluster.by="de pattern"), it implements a row standardization that takes account of NA values and standard deviations that might be zero.

coolmap sets the following heatmap.2 arguments internally: Rowv, Colv, scale, density.info, trace, col, symbreaks, symkey, dendrogram, key.title and key.xlab. These arguments are therefore reserved and cannot be varied. Other than these reserved arguments, any other heatmap.2 argument can be included in the coolmap call, thereby giving full access to heatmap.2 functionality.

Value

A plot is created on the current graphics device. A list is also invisibly returned, see heatmap.2 for details.

Author(s)

Gordon Smyth

See Also

heatmap.2, hclust, dist.

An overview of diagnostic functions available in LIMMA is given in 09.Diagnostics.

Examples

# Simulate gene expression data for 50 genes and 6 microarrays.
# Samples are in two groups
# First 50 probes are differentially expressed in second group
ngenes <- 50
sd <- 0.3*sqrt(4/rchisq(ngenes,df=4))
x <- matrix(rnorm(ngenes*6,sd=sd),ngenes,6)
rownames(x) <- paste("Gene",1:ngenes)
x <- x + seq(from=0, to=16, length=ngenes)
x[,4:6] <- x[,4:6] + 2
coolmap(x)

Description

Test whether the leading members of ordered lists significantly overlap.

Usage

cumOverlap(ol1, ol2)

Arguments

Details

The function compares the top n members of each list, for every possible n, and conducts an hypergeometric test for overlap. The function returns the value of n giving the smallest p-value.

The p-values are adjusted for multiple testing in a similar way to Bonferroni's method, but starting from the top of th e ranked list instead of from the smallest p-values. This approach is designed to be sensitive to contexts where the number of Ids involved in the significant overlap are a small proportion of the total.

The vectors ol1 and ol2 do not need to be of the same length, but only values in common between the two vectors will be used in the calculation.

This method was described in Chapter 4 of Wu (2011).

Value

List containing the following components:

Author(s)

Gordon Smyth and Di Wu

References

Wu, D (2011). Finding hidden relationships between gene expression profiles with application to breast cancer biology. PhD thesis, University of Melbourne. http://hdl.handle.net/11343/36278

Examples

GeneIds <- paste0("Gene",1:50)
ol1 <- GeneIds
ol2 <- c(sample(GeneIds[1:5]), sample(GeneIds[6:50]))
coa <- cumOverlap(ol1, ol2)
coa$p.min
coa$id.overlap

Description

Identify which genes are significantly differentially expressed for each contrast from a fit object containing p-values and test statistics. A number of different multiple testing strategies are offered that adjust for multiple testing down the genes as well as across contrasts for each gene.

Usage

## S3 method for class 'MArrayLM'
decideTests(object, method = "separate", adjust.method = "BH", p.value = 0.05,
            lfc = 0, ...)
## Default S3 method:
decideTests(object, method = "separate", adjust.method = "BH", p.value = 0.05,
            lfc = 0, coefficients = NULL, cor.matrix = NULL, tstat = NULL, df = Inf,
            genewise.p.value = NULL, ...)

Arguments

Details

This function can be applied to a matrix of p-values but is more often applied to an MArrayLM fit object produced by eBayes or treat. In either case, rows of object correspond to genes and columns to coefficients or contrasts.

This function applies a multiple testing procedure and a significance level cutoff to the statistics contained in object. It implements a number of multiple testing procedures for determining whether each statistic should be considered significantly different from zero.

method="separate" will apply multiple testing adjustments to each column of p-values separately. Setting method="separate" is equivalent to using topTable separately for each coefficient in the linear model fit and will identify the same probes as significantly differentially expressed if adjust.method is the same. method="global" will treat the entire matrix of t-statistics as a single vector of unrelated tests. method="hierarchical" adjusts down genes and then across contrasts. method="nestedF" adjusts down genes according to overall F-tests and then uses classifyTestsF to classify contrasts as significant or not for the selected genes.

The default method="separate" and adjust.method="BH" settings are appropriate for most analyses. method="global" is useful when it is important that the same t-statistic cutoff should correspond to statistical significance for all the contrasts. The "nestedF" method was proposed by Michaud et al (2008) and achieves better consistency between contrasts than the other methods. It provides formal error rate control at the gene level but not for individual contrasts. See the classifyTestsF help page for more detail about the "nestedF" method.

If object is a MArrayLM linear model fit, then the "hierarchical" method conducts row-wise F-tests and then proceeds to t-tests for those rows with significant F-tests. The multiple testing adjustment is applied initially to the F-tests and then, with an adjusted level, to the t-tests for each significant row.

Also see the limma User's Guide for a discussion of the statistical properties of the various adjustment methods.

Value

An object of class TestResults. This is essentially a numeric matrix with elements -1, 0 or 1 depending on whether each t-statistic is classified as significantly negative, not significant or significantly positive.

If lfc>0 then contrasts are judged significant only when the log2-fold change is at least this large in absolute value. For example, one might choose lfc=log2(1.5) to restrict to 50% changes or lfc=1 for 2-fold changes. In this case, contrasts must satisfy both the p-value and the fold-change cutoff to be judged significant.

Note

Although this function enables users to set p-value and lfc cutoffs simultaneously, this combination criterion is not recommended. logFC cutoffs tend to favor low expressed genes and thereby reduce rather than increase biological significance. Unless the fold changes and p-values are very highly correlated, the addition of a fold change cutoff can increase the family-wise error rate or false discovery rate above the nominal level. Users wanting to use fold change thresholding are recommended to use treat instead of eBayes and to leave lfc at the default value when using decideTests.

Author(s)

Gordon Smyth

References

Michaud J, Simpson KM, Escher R, Buchet-Poyau K, Beissbarth T, Carmichael C, Ritchie ME, Schutz F, Cannon P, Liu M, Shen X, Ito Y, Raskind WH, Horwitz MS, Osato M, Turner DR, Speed TP, Kavallaris M, Smyth GK, Scott HS (2008). Integrative analysis of RUNX1 downstream pathways and target genes. BMC Genomics 9, 363. doi:10.1186/1471-2164-9-363

See Also

An overview of multiple testing functions is given in 08.Tests.

Description

Convert a design matrix in terms of individual channels to ones in terms of M-values or A-values for two-color microarray data.

Usage

designI2M(design)
designI2A(design)

Arguments

Details

If design is a model matrix suitable for modelling individual log-intensities for two color microarray data, then designI2M computes the corresponding model matrix for modelling M-values (log-ratios) and designI2A computes the model matrix for modelling A-values (average log-intensities).

Note that the matrices designI2M(design) or designI2A(design) may be singular if not all of the coefficients are estimable from the M or A-values. In that case there will be columns containing entirely zeros.

Value

numeric model matrix with half as many rows as design

Author(s)

Gordon Smyth

See Also

model.matrix in the stats package.

An overview of individual channel linear model functions in limma is given by 07.SingleChannel.

Examples

X <- cbind(1,c(1,1,1,1,0,0,0,0),c(0,0,0,0,1,1,1,1))
designI2M(X)
designI2A(X)

Description

Compute the proportion of negative controls greater than each observed expression value. Particularly useful for Illumina BeadChips.

Usage

## S3 method for class 'EListRaw'
detectionPValues(x, status = NULL, ...)
## Default S3 method:
detectionPValues(x, status, negctrl = "negative", ...)

Arguments

Details

The rows of x for which status == negctrl are assumed to correspond to negative control probes.

For each column of x, the detection p-values are defined as (N.eq/2 + N.gt) / N.neg, where N.gt is the number of negative controls with expression greater than the observed value, N.eq is the number of negative controls with expression equal to the observed value, and N.neg is the total number of negative controls.

When used on Illumina BeadChip data, this function produces essentially the same detection p-values as returned by Illumina's GenomeStudio software.

Value

numeric matrix of same dimensions as x containing detection p-values.

Author(s)

Gordon Smyth

References

Shi W, de Graaf C, Kinkel S, Achtman A, Baldwin T, Schofield L, Scott H, Hilton D, Smyth GK (2010). Estimating the proportion of microarray probes expressed in an RNA sample. Nucleic Acids Research 38(7), 2168-2176. doi:10.1093/nar/gkp1204

See Also

An overview of LIMMA functions to read expression data is given in 03.ReadingData.

read.idat reads Illumina BeadChip expression data from binary IDAT files.

neqc performs normexp background correction and quantile normalization aided by control probes.

Examples

## Not run: 
# Read Illumina binary IDAT files
x <- read.idat(idat, bgx)
x$other$Detection <- detectionPValues(x)
y <- neqc(x)

## End(Not run)

Description

Given a linear model fit at the exon level, test for differences in exon retention between experimental conditions.

Usage

diffSplice(fit, geneid, exonid=NULL, robust=FALSE, verbose=TRUE)

Arguments

Details

This function tests for differential exon usage for each gene and for each column of fit.

Testing for differential exon usage is equivalent to testing whether the log-fold-changes in the fit differ between exons for the same gene. Two different tests are provided. The first is an F-test for differences between the log-fold-changes. The other is a series of t-tests in which each exon is compared to the average of all other exons for the same gene. The exon-level t-tests are converted into a genewise test by adjusting the p-values for the same gene by Simes method. The minimum adjusted p-value is then used for each gene.

This function can be used on data from an exon microarray or can be used in conjunction with voom for exon-level RNA-seq counts.

Value

An object of class MArrayLM containing both exon level and gene level tests. Results are sorted by geneid and by exonid within gene.

Author(s)

Gordon Smyth and Charity Law

See Also

topSplice, plotSplice

A summary of functions available in LIMMA for RNA-seq analysis is given in 11.RNAseq.

Examples

## Not run: 
v <- voom(dge,design)
fit <- lmFit(v,design)
ex <- diffSplice(fit,geneid="EntrezID")
topSplice(ex)
plotSplice(ex)

## End(Not run)

Description

Retrieve the number of rows (genes) and columns (arrays) for an RGList, MAList or MArrayLM object.

Usage

## S3 method for class 'RGList'
dim(x)

Arguments

Details

Microarray data objects share many analogies with ordinary matrices in which the rows correspond to spots or genes and the columns to arrays. These methods allow one to extract the size of microarray data objects in the same way that one would do for ordinary matrices.

A consequence is that row and column commands nrow(x), ncol(x) and so on also work.

Value

Numeric vector of length 2. The first element is the number of rows (genes) and the second is the number of columns (arrays).

Author(s)

Gordon Smyth

See Also

dim in the base package.

02.Classes gives an overview of data classes used in LIMMA.

Examples

M <- A <- matrix(11:14,4,2)
rownames(M) <- rownames(A) <- c("a","b","c","d")
colnames(M) <- colnames(A) <- c("A1","A2")
MA <- new("MAList",list(M=M,A=A))
dim(M)
ncol(M)
nrow(M)

Description

Retrieve the dimension names of a microarray data object.

Usage

## S3 method for class 'RGList'
dimnames(x)
## S3 replacement method for class 'RGList'
dimnames(x) <- value

Arguments

Details

The dimension names of a microarray object are the same as those of the most important matrix component of that object.

A consequence is that rownames and colnames will work as expected.

Value

Either NULL or a list of length 2. If a list, its components are either NULL or a character vector the length of the appropriate dimension of x.

Author(s)

Gordon Smyth

See Also

dimnames in the base package.

02.Classes gives an overview of data classes used in LIMMA.

Description

Estimate the intra-block correlation given a block structure for the arrays or samples.

Usage

duplicateCorrelation(object, design=NULL, ndups=2, spacing=1, block=NULL,
                     trim=0.15, weights=NULL)

Arguments

Details

When block=NULL, this function estimates the correlation between duplicate spots (regularly spaced within-array replicate spots). If block is not null, this function estimates the correlation between repeated observations on the blocking variable. Typically the blocks are biological replicates and repeated observations on the same block may be correlated. In either case, the correlation is estimated by fitting a mixed linear model by REML individually for each gene. The function also returns a consensus correlation, which is a robust average of the individual correlations, intended for input to functions such as lmFit, gls.series or voom.

It is not possible to estimate correlations between duplicate spots and with sample blocks simultaneously. If block is not null, then the function will set ndups=1, which is equivalent to ignoring duplicate spots.

For this function to return statistically useful results, there must be at least two more arrays than the number of coefficients to be estimated, i.e., two more than the column rank of design.

The function may take long time to execute as it fits a mixed linear model for each gene using an iterative algorithm.

If present, ndups and spacing will be extracted from object$printer$ndups and object$printer$spacing.

Value

A list with components

Author(s)

Gordon Smyth

References

Smyth, G. K., Michaud, J., and Scott, H. (2005). The use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 21(9), 2067-2075. [http://bioinformatics.oxfordjournals.org/content/21/9/2067] [Preprint with corrections: https://gksmyth.github.io/pubs/dupcor.pdf]

See Also

These functions use mixedModel2Fit from the statmod package.

An overview of linear model functions in limma is given by 06.LinearModels.

Examples

# Simulate a paired experiment with incomplete blocks
Block <- c(1,1,2,2,3,3,4,4,5,6,7,8)
Treat <- factor(c(1,2,1,2,1,2,1,2,1,2,1,2))
design <- model.matrix(~Treat)

ngenes <- 50
nsamples <- 12
y <- matrix(rnorm(ngenes*nsamples),ngenes,nsamples)
rownames(y) <- paste0("Gene",1:ngenes)

# Estimate the within-block correlation
dupcor <- duplicateCorrelation(y,design,block=Block)
dupcor$consensus.correlation

# Estimate the treatment effect using both complete and incomplete blocks
fit <- lmFit(y,design,block=Block,correlation=dupcor$consensus)
fit <- eBayes(fit)
topTable(fit,coef=2)

Description

Given a linear model fit from lmFit, compute moderated t-statistics, moderated F-statistic, and log-odds of differential expression by empirical Bayes moderation of the standard errors towards a global value.

Usage

eBayes(fit, proportion = 0.01, stdev.coef.lim = c(0.1,4),
       trend = FALSE, robust = FALSE, winsor.tail.p = c(0.05,0.1))
treat(fit, fc = 1.2, lfc = NULL, trend = FALSE,
      robust = FALSE, winsor.tail.p = c(0.05,0.1))

Arguments

Details

These functions are used to rank genes in order of evidence for differential expression. They use an empirical Bayes method to squeeze the genewise-wise residual variances towards a common value (or towards a global trend) (Smyth, 2004; Phipson et al, 2016). The degrees of freedom for the individual variances are increased to reflect the extra information gained from the empirical Bayes moderation, resulting in increased statistical power to detect differential expression.

Theese functions accept as input an MArrayLM fitted model object fit produced by lmFit. The columns of fit define a set of contrasts which are to be tested equal to zero. The fitted model object may have been processed by contrasts.fit before being passed to eBayes to convert the coefficients of the original design matrix into an arbitrary number of contrasts.

The empirical Bayes moderated t-statistics test each individual contrast equal to zero. For each gene (row), the moderated F-statistic tests whether all the contrasts are zero. The F-statistic is an overall test computed from the set of t-statistics for that probe. This is exactly analogous the relationship between t-tests and F-statistics in conventional anova, except that the residual mean squares have been moderated between genes.

The estimates s2.prior and df.prior are computed by fitFDist. s2.post is the weighted average of s2.prior and sigma^2 with weights proportional to df.prior and df.residual respectively. The log-odds of differential expression lods was called the B-statistic by Loennstedt and Speed (2002). The F-statistics F are computed by classifyTestsF with fstat.only=TRUE.

eBayes does not compute ordinary t-statistics because they always have worse performance than the moderated versions. The ordinary (unmoderated) t-statistics can, however, can be easily extracted from the linear model output for comparison purposes—see the example code below.

treat computes empirical Bayes moderated-t p-values relative to a minimum fold-change threshold. Instead of testing for genes that have true log-fold-changes different from zero, it tests whether the true log2-fold-change is greater than lfc in absolute value (McCarthy and Smyth, 2009). In other words, it uses an interval null hypothesis, where the interval is [-lfc,lfc]. When the number of DE genes is large, treat is often useful for giving preference to larger fold-changes and for prioritizing genes that are biologically important. treat is concerned with p-values rather than posterior odds, so it does not compute the B-statistic lods. The idea of thresholding doesn't apply to F-statistics in a straightforward way, so moderated F-statistics are also not computed. When fc=1 and lfc=0, treat is identical to eBayes, except that F-statistics and B-statistics are not computed. The fc threshold is usually chosen relatively small, because genes need to have fold changes substantially greater than the testing threshold in order to be considered statistically significant. Typical values for fc are 1.1, 1.2 or 1.5. The top genes chosen by treat can be examined using topTreat.

The treat threshold can be specified either as a fold-change via fc or as a log2-fold-change via lfc, with lfc = log2(fc). Note that the treat testing procedure is considerably more rigorous and conservative than simply applying same fc values as a fold-change cutoff to the list of differentially expressed genes. Indeed, the observed log2-fold-change needs to substantially larger than lfc for a gene to be called as statistically significant by treat. The threshold should be chosen as a small value below which results should be ignored rather than as a target fold-change. In practice, modest values for fc such as 1.1, 1.2 or 1.5 are usually the most useful. Setting fc=1.2 or fc=1.5 will usually cause most differentially expressed genes to have estimated fold-changes of 2-fold or greater, depending on the sample size and precision of the experiment. Larger thresholds are usually overly conservative and counter productive. In general, the fc threshold should be chosen sufficiently small so that a worthwhile number of DE genes remain, otherwise the purpose of prioritizing genes with larger fold-changes will be defeated.

The use of eBayes or treat with trend=TRUE is known as the limma-trend method (Law et al, 2014; Phipson et al, 2016). With this option, an intensity-dependent trend is fitted to the prior variances s2.prior. Specifically, squeezeVar is called with the covariate equal to Amean, the average log2-intensity for each gene. The trend that is fitted can be examined by plotSA. limma-trend is useful for processing expression values that show a mean-variance relationship. This is often useful for microarray data, and it can also be applied to RNA-seq counts that have been converted to log2-counts per million (logCPM) values (Law et al, 2014). When applied to RNA-seq logCPM values, limma-trend give similar results to the voom method. The voom method incorporates the mean-variance trend into the precision weights, whereas limma-trend incorporates the trend into the empirical Bayes moderation. limma-trend is somewhat simpler than voom because it assumes that the sequencing depths (library sizes) are not wildly different between the samples and it applies the mean-variance trend on a genewise basis instead to individual observations. limma-trend is recommended for RNA-seq analysis when the library sizes are reasonably consistent (less than 3-fold difference from smallest to largest) because of its simplicity and speed.

If robust=TRUE then the robust empirical Bayes procedure of Phipson et al (2016) is used. This is frequently useful to protect the empirical Bayes procedure against hyper-variable or hypo-variable genes, especially when analysing RNA-seq data. See squeezeVar for more details.

Value

eBayes produces an object of class MArrayLM (see MArrayLM-class) containing everything found in fit plus the following added components:

The matrices t, p.value and lods have the same dimensions as the input object fit, with rows corresponding to genes and columns to coefficients or contrasts. The vectors s2.prior, df.prior, df.total, F and F.p.value correspond to rows, with length equal to the number of genes. The vector var.prior corresponds to columns, with length equal to the number of contrasts. If s2.prior or df.prior have length 1, then the same value applies to all genes.

s2.prior, df.prior and var.prior contain empirical Bayes hyperparameters used to obtain df.total, s2.post and lods.

treat a produces an MArrayLM object similar to that from eBayes but without lods, var.prior, F or F.p.value.

Note

The algorithm used by eBayes and treat with robust=TRUE was revised slightly in limma 3.27.6. The minimum df.prior returned may be slightly smaller than previously.

Author(s)

Gordon Smyth and Davis McCarthy

References

Law CW, Chen Y, Shi W, Smyth GK (2014). Voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biology 15, R29. doi:10.1186/gb-2014-15-2-r29. See also the Preprint Version at https://gksmyth.github.io/pubs/VoomPreprint.pdf incorporating some notational corrections.

Loennstedt I, and Speed TP (2002). Replicated microarray data. Statistica Sinica 12, 31-46.

McCarthy D J, Smyth GK (2009). Testing significance relative to a fold-change threshold is a TREAT. Bioinformatics 25, 765-771. doi:10.1093/bioinformatics/btp053

Phipson B, Lee S, Majewski IJ, Alexander WS, Smyth GK (2016). Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Annals of Applied Statistics 10, 946-963. doi:10.1214/16-AOAS920

Smyth GK (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology Volume 3, Issue 1, Article 3. doi:10.2202/1544-6115.1027. See also the Preprint Version https://gksmyth.github.io/pubs/ebayes.pdf incorporating corrections to 30 June 2009.

See Also

squeezeVar, fitFDist, tmixture.matrix, plotSA.

An overview of linear model functions in limma is given by 06.LinearModels.

Examples

#  See also lmFit examples

#  Simulate gene expression data,
#  6 microarrays and 100 genes with one gene differentially expressed
set.seed(2016)
sigma2 <- 0.05 / rchisq(100, df=10) * 10
y <- matrix(rnorm(100*6,sd=sqrt(sigma2)),100,6)
design <- cbind(Intercept=1,Group=c(0,0,0,1,1,1))
y[1,4:6] <- y[1,4:6] + 1
fit <- lmFit(y,design)

#  Moderated t-statistic
fit <- eBayes(fit)
topTable(fit,coef=2)

#  Ordinary t-statistic
ordinary.t <- fit$coef[,2] / fit$stdev.unscaled[,2] / fit$sigma

# Treat relative to a 10% fold-change
tfit <- treat(fit, fc=1.1)
topTreat(tfit,coef=2)

Description

A list-based S4 classes for storing expression values (E-values), for example for a set of one-channel microarrays or a set of RNA-seq samples. EListRaw holds expression values on the raw scale. EList holds expression values on the log scale, usually after background correction and normalization.

EListRaw objects are often created by read.maimages, while EList objects are often created by normalizeBetweenArrays or by voom. Alternatively, an EList object can be created directly by new("EList",x), where x is a list.

Required Components

These classes contains no slots (other than .Data), but objects should contain a list component E:

E

numeric matrix containing expression values. In an EListRaw object, the expression values are unlogged, while in an EList object, they are log2 values. Rows correspond to probes and columns to samples.

Optional Components

Optional components include:

Eb

numeric matrix containing unlogged background expression values, of same dimensions as E. For an EListRaw object only.

weights

numeric matrix of same dimensions as E containing relative spot quality weights. Elements should be non-negative.

other

list containing other matrices, all of the same dimensions as E.

genes

data.frame containing probe information. Should have one row for each probe. May have any number of columns.

targets

data.frame containing information on the target RNA samples. Rows correspond to samples. May have any number of columns.

Valid EList or EListRaw objects may contain other optional components, but all probe or sample information should be contained in the above components.

Methods

These classes inherit directly from class list so any operation appropriate for lists will work on objects of this class. In addition, EList objects can be subsetted and combined. EList objects will return dimensions and hence functions such as dim, nrow and ncol are defined. ELists also inherit a show method from the virtual class LargeDataObject, which means that ELists will print in a compact way.

Author(s)

Gordon Smyth

See Also

02.Classes gives an overview of all the classes defined by this package.

ExpressionSet is a more formal class in the Biobase package used for the same purpose.

Examples

# Two ways to make an EList object:

y <- matrix(rnorm(10,5),10,5)
rownames(y) <- paste0("Gene",1:10)
colnames(y) <- LETTERS[1:5]
Genes <- data.frame(Chr=sample(1:21,10))
row.names(Genes) <- row.names(y)

# Create the object, than add components:
E <- new("EList")
E$E <- y
E$genes <- Genes

# Create with components:
E <- new("EList", list(E=y, genes=Genes))

Description

Extract the matrix of log-expression values from an MAList object.

Usage

exprs.MA(MA)

Arguments

Details

Converts M and A-values to log-expression values. The output matrix will have two columns for each array, in the order green, red for each array.

This contrasts with as.matrix.MAList which extracts the M-values only, or RG.MA which converts to expression values in RGList form.

Value

A numeric matrix with twice the columns of the input.

Author(s)

Gordon Smyth

See Also

02.Classes gives an overview of data classes used in LIMMA.

Description

Moment estimation of the parameters of a scaled F-distribution given one of the degrees of freedom. This function is called internally by eBayes and squeezeVar and is not usually called directly by a user.

Usage

fitFDist(x, df1, covariate=NULL)
fitFDistRobustly(x, df1, covariate=NULL, winsor.tail.p=c(0.05,0.1), trace=FALSE)

Arguments

Details

fitFDist implements an algorithm proposed by Smyth (2004) and Phipson et al (2016). It estimates scale and df2 under the assumption that x is distributed as scale times an F-distributed random variable on df1 and df2 degrees of freedom. The parameters are estimated using the method of moments, specifically from the mean and variance of the x values on the log-scale.

When covariate is supplied, a spline curve trend will be estimated for the x values and the estimation will be adjusted for this trend (Phipson et al, 2016).

fitFDistRobustly is similar to fitFDist except that it computes the moments of the Winsorized values of x, making it robust against left and right outliers. Larger values for winsor.tail.p produce more robustness but less efficiency. When covariate is supplied, a loess trend is estimated for the x values. The robust method is described by Phipson et al (2016).

As well as estimating the F-distribution for the bulk of the cases, i.e., with outliers discounted, fitFDistRobustly also returns an estimated F-distribution with reduced df2 that might be appropriate for each outlier case.

Value

fitFDist produces a list with the following components:

fitFDistRobustly returns the following components as well:

Note

The algorithm used by fitFDistRobustly was revised slightly in limma 3.27.6. The prob.outlier value, which is the lower bound for df2.shrunk, may be slightly smaller than previously.

Author(s)

Gordon Smyth and Belinda Phipson

References

Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology Volume 3, Issue 1, Article 3. doi:10.2202/1544-6115.1027 https://gksmyth.github.io/pubs/ebayes.pdf

Phipson, B, Lee, S, Majewski, IJ, Alexander, WS, and Smyth, GK (2016). Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Annals of Applied Statistics 10, 946-963. doi:10.1214/16-AOAS920

See Also

This function is called by squeezeVar, which in turn is called by eBayes and treat.

This function calls trigammaInverse.

Examples

x <- rf(100,df1=8,df2=16)
fitFDist(x,df1=8)

Description

Fit Intercept to Vector of Gamma Distributed Variates

Usage

fitGammaIntercept(y,offset=0,maxit=1000)

Arguments

Details

The values y are assumed to follow a gamma distribution with common shape parameter and with expected values given by x+offset. The function implements a globally convergent Newton iteration to estimate x.

Value

Numeric value giving intercept.

Author(s)

Gordon Smyth and Belinda Phipson

References

Phipson, B. (2013). Empirical Bayes modelling of expression profiles and their associations. PhD Thesis. University of Melbourne, Australia.

See Also

This function is called by genas.

Examples

offset <- runif(10)
x <- 9
mu <- x+offset
y <- rgamma(10,shape=20,scale=mu/20)
fitGammaIntercept(y,offset=offset)

Description

Fit Mixture Model by Non-Linear Least Squares

Usage

fitmixture(log2e, mixprop, niter = 4, trace = FALSE)

Arguments

Details

A mixture experiment is one in which two reference RNA sources are mixed in different proportions to create experimental samples. Mixture experiments have been used to evaluate genomic technologies and analysis methods (Holloway et al, 2006). This function uses all the data for each gene to estimate the expression level of the gene in each of two pure samples.

The function fits a nonlinear mixture model to the log2 expression values for each gene. The expected values of log2e for each gene are assumed to be of the form log2( mixprop*Y1 + (1-mixprop)*Y2 ) where Y1 and Y2 are the expression levels of the gene in the two reference samples being mixed. The mixprop values are the same for each gene but Y1 and Y2 are specific to the gene. The function returns the estimated values A=0.5*log2(Y1*Y2) and M=log2(Y2/Y1) for each gene.

The nonlinear estimation algorithm implemented in fitmixture uses a nested Gauss-Newton iteration (Smyth, 1996). It is fully vectorized so that the estimation is done for all genes simultaneously.

Value

List with three components:

Author(s)

Gordon K Smyth

References

Holloway, A. J., Oshlack, A., Diyagama, D. S., Bowtell, D. D. L., and Smyth, G. K. (2006). Statistical analysis of an RNA titration series evaluates microarray precision and sensitivity on a whole-array basis. BMC Bioinformatics 7, Article 511. doi:10.1186/1471-2105-7-511

Smyth, G. K. (1996). Partitioned algorithms for maximum likelihood and other nonlinear estimation. Statistics and Computing, 6, 201-216. https://gksmyth.github.io/pubs/partitio.pdf

Examples

ngenes <- 100
TrueY1 <- rexp(ngenes)
TrueY2 <- rexp(ngenes)
mixprop <- matrix(c(0,0.25,0.75,1),1,4)
TrueExpr <- TrueY1 

log2e <- log2(TrueExpr) + matrix(rnorm(ngenes*4),ngenes,4)*0.1
out <- fitmixture(log2e,mixprop)

# Plot true vs estimated log-ratios
plot(log2(TrueY1/TrueY2), out$M)

Description

Obtains fitted values from a fitted microarray linear model object.

Usage

## S3 method for class 'MArrayLM'
fitted(object, ...)

Arguments

Value

A numeric matrix of fitted values.

Author(s)

Gordon Smyth

See Also

fitted

Description

Calculates biological correlation between two gene expression profiles.

Usage

genas(fit, coef=c(1,2), subset="all", plot=FALSE, alpha=0.4)

Arguments

Details

The function estimates the biological correlation between two different contrasts in a linear model. By biological correlation, we mean the correlation that would exist between the log2-fold changes (logFC) for the two contrasts, if measurement error could be eliminated and the true log-fold-changes were known. This function is motivated by the fact that different contrasts for a linear model are often strongly correlated in a technical sense. For example, the estimated logFC for multiple treatment conditions compared back to the same control group will be positively correlated even in the absence of any biological effect. This function aims to separate the biological from the technical components of the correlation. The method is explained briefly in Majewski et al (2010) and in full detail in Phipson (2013).

The subset argument specifies whether and how the fit object should be subsetted. Ideally, only genes that are truly differentially expressed for one or both of the contrasts should be used estimate the biological correlation. The default is "all", which uses all genes in the fit object to estimate the biological correlation. The option "Fpval" chooses genes based on how many F-test p-values are estimated to be truly significant using the function propTrueNull. This should capture genes that display any evidence of differential expression in either of the two contrasts. The options "p.union" and "p.int" are based on the moderated t p-values from both contrasts. From the propTrueNull function an estimate of the number of p-values truly significant in either of the two contrasts can be obtained. "p.union" takes the union of these genes and "p.int" takes the intersection of these genes. The other options, "logFC" and "predFC" subsets on genes that attain a logFC or predFC at least as large as the 90th percentile of the log fold changes or predictive log fold changes on the absolute scale.

The plot option is a logical argument that specifies whether or not to plot a scatter plot of log-fold-changes for the two contrasts. The biological and technical correlations are overlaid on the scatterplot using semi-transparent ellipses. library(ellipse) is required to enable the plotting of ellipses.

Value

genas produces a list with the following components:

Note

As present, genas assumes that technical correlations between coefficients are the same for all genes, and hence it only works with fit objects that were created without observation weights or missing values. It does not work with voom pipelines, because these involve observation weights.

Author(s)

Belinda Phipson and Gordon Smyth

References

Majewski, IJ, Ritchie, ME, Phipson, B, Corbin, J, Pakusch, M, Ebert, A, Busslinger, M, Koseki, H, Hu, Y, Smyth, GK, Alexander, WS, Hilton, DJ, and Blewitt, ME (2010). Opposing roles of polycomb repressive complexes in hematopoietic stem and progenitor cells. Blood 116, 731-739. http://www.bloodjournal.org/content/116/5/731

Phipson, B. (2013). Empirical Bayes modelling of expression profiles and their associations. PhD Thesis. University of Melbourne, Australia. http://hdl.handle.net/11343/38162

Ritchie, ME, Phipson, B, Wu, D, Hu, Y, Law, CW, Shi, W, and Smyth, GK (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Research 43, e47. http://nar.oxfordjournals.org/content/43/7/e47

See Also

lmFit, eBayes, contrasts.fit

Examples

#  Simulate gene expression data

#  Three conditions (Control, A and B) and 1000 genes
ngene <- 1000
mu.A <- mu.B <- mu.ctrl <- rep(5,ngene)

#  200 genes are differentially expressed.
#  All are up in condition A and down in B
#  so the biological correlation is negative.
mu.A[1:200] <- mu.ctrl[1:200]+2
mu.B[1:200] <- mu.ctrl[1:200]-2

#  Two microarrays for each condition
mu <- cbind(mu.ctrl,mu.ctrl,mu.A,mu.A,mu.B,mu.B)
y <- matrix(rnorm(6000,mean=mu,sd=1),ngene,6)

# two experimental groups and one control group with two replicates each
group <- factor(c("Ctrl","Ctrl","A","A","B","B"), levels=c("Ctrl","A","B"))
design <- model.matrix(~group)

# fit a linear model
fit <- lmFit(y,design)
fit <- eBayes(fit)

#  Estimate biological correlation between the logFC profiles
#  for A-vs-Ctrl and B-vs-Ctrl
genas(fit, coef=c(2,3), plot=TRUE, subset="F")

Description

Test whether a set of genes is highly ranked relative to other genes in terms of a given statistic. Genes are assumed to be independent.

Usage

geneSetTest(index, statistics, alternative = "mixed", type= "auto",
            ranks.only = TRUE, nsim=9999)
wilcoxGST(index, statistics, ...)

Arguments

Details

These functions compute a p-value to test the hypothesis that the indexed test set of genes tends to be more highly ranked in terms of some test statistic compared to randomly chosen genes. The statistic might be any statistic of interest, for example a t-statistic or F-statistic for differential expression. Like all gene set tests, these functions can be used to detect differential expression for a group of genes, even when the effects are too small or there is too little data to detect the genes individually.

wilcoxGST is a synonym for geneSetTest with ranks.only=TRUE. This version of the test procedure was developed by Michaud et al (2008), who called it mean-rank gene-set enrichment.

geneSetTest performs a competitive test in the sense that genes in the test set are compared to other genes (Goeman and Buhlmann, 2007). If the statistic is a genewise test statistic for differential expression, then geneSetTest tests whether genes in the set are more differentially expressed than genes not in the set. By contrast, a self-contained gene set test such as roast tests whether genes in the test set are differentially expressed, in an absolute sense, without regard to any other genes on the array.

Because it is based on permuting genes, geneSetTest assumes that the different genes (or probes) are statistically independent. (Strictly speaking, it assumes that the genes in the set are no more correlated on average than randomly chosen genes.) If inter-gene correlations are present, then a statistically significant result from geneSetTest indicates either that the set is highly ranked or that the genes in the set are positively correlated on average (Wu and Smyth, 2012). Unless gene sets with positive correlations are particularly of interest, it may be advisable to use camera or cameraPR instead to adjust the test for inter-gene correlations. Inter-gene correlations are likely to be present in differential expression experiments with biologically heterogeneous experimental units. On the other hand, the assumption of independence between genes should hold when the replicates are purely technical, i.e., when there is no biological variability between the replicate arrays in each experimental condition.

The statistics are usually a set of probe-wise statistics arising for some comparison from a microarray experiment. They may be t-statistics, meaning that the genewise null hypotheses would be rejected for large positive or negative values, or they may be F-statistics, meaning that only large values are significant. Any set of signed statistics, such as log-ratios, M-values or moderated t-statistics, are treated as t-like. Any set of unsigned statistics, such as F-statistics, posterior probabilities or chi-square tests are treated as F-like. If type="auto" then the statistics will be taken to be t-like if they take both positive and negative values and will be taken to be F-like if they are all of the same sign.

There are four possible alternatives to test for. alternative=="up" means the genes in the set tend to be up-regulated, with positive t-statistics. alternative=="down" means the genes in the set tend to be down-regulated, with negative t-statistics. alternative=="either" means the set is either up or down-regulated as a whole. alternative=="mixed" test whether the genes in the set tend to be differentially expressed, without regard for direction. In this case, the test will be significant if the set contains mostly large test statistics, even if some are positive and some are negative.

The latter three alternatives are appropriate when there is a prior expection that all the genes in the set will react in the same direction. The "mixed" alternative is appropriate if you know only that the genes are involved in the relevant pathways, possibly in different directions. The "mixed" is the only meaningful alternative with F-like statistics.

The test statistic used for the gene-set-test is the mean of the statistics in the set. If ranks.only is TRUE the only the ranks of the statistics are used. In this case the p-value is obtained from a Wilcoxon test. If ranks.only is FALSE, then the p-value is obtained by simulation using nsim random sets of genes.

Value

numeric value giving the estimated p-value.

Note

Wu and Smyth (2012) show that geneSetTest does not does correct for inter-gene correlations and is more likely to assign small p-values to sets containing positive correlated genes. The function cameraPR is recommended as a alternative.

Author(s)

Gordon Smyth and Di Wu

References

Wu, D, and Smyth, GK (2012). Camera: a competitive gene set test accounting for inter-gene correlation. Nucleic Acids Research 40(17), e133. doi:10.1093/nar/gks461

Goeman, JJ, and Buhlmann P (2007). Analyzing gene expression data in terms of gene sets: methodological issues. Bioinformatics 23, 980-987.

Michaud, J, Simpson, KM, Escher, R, Buchet-Poyau, K, Beissbarth, T, Carmichael, C, Ritchie, ME, Schutz, F, Cannon, P, Liu, M, Shen, X, Ito, Y, Raskind, WH, Horwitz, MS, Osato, M, Turner, DR, Speed, TP, Kavallaris, M, Smyth, GK, and Scott, HS (2008). Integrative analysis of RUNX1 downstream pathways and target genes. BMC Genomics 9, 363. doi:10.1186/1471-2164-9-363

See Also

cameraPR, camera, roast, barcodeplot, wilcox.test.

There is a topic page on 10.GeneSetTests.

Examples

stat <- rnorm(100)
sel <- 1:10; stat[sel] <- stat[sel]+1
wilcoxGST(sel,stat)

Description

Given an expression data object of any known class, get the expression values, weights, probe annotation and A-values that are needed for linear modelling. This function is called by the linear modelling functions in LIMMA.

Usage

getEAWP(object)

Arguments

Details

Rows correspond to probes and columns to RNA samples.

In the case of two-color microarray data objects (MAList or marrayNorm), Amean is the vector of row means of the matrix of A-values. For other data objects, Amean is the vector of row means of the matrix of expression values.

From April 2013, the rownames of the output exprs matrix are required to be unique. If object has no row names, then the output rownames of exprs are 1:nrow(object). If object has row names but with duplicated names, then the rownames of exprs are set to 1:nrow(object) and the original row names are preserved in the ID column of probes.

object should be a normalized data object. getEAWP will return an error if object is a non-normalized data object such as RGList or EListRaw, because these do not contain log-expression values.

Value

A list with components

exprs is the only required component. The other components will be NULL if not found in the input object.

Author(s)

Gordon Smyth

See Also

02.Classes gives an overview of data classes used in LIMMA.

Description

From the Block, Row and Column information in a genelist, determine the number of grid rows and columns on the array and the number of spot rows and columns within each grid.

Usage

getLayout(gal, guessdups=FALSE)
getLayout2(galfile)
getDupSpacing(ID)

Arguments

Details

A GenePix Array List (GAL) file is a list of genes and associated information produced by an Axon microarray scanner. The function getLayout determines the print layout from a data frame created from a GAL file or gene list. The data.frame must contain columns Block, Column and Row. (The number of tip columns is assumed to be either one or four.)

On some arrays, each probe may be duplicated a number of times (ndups) at regular intervals (spacing) in the GAL file. getDupSpacing determines valid values for ndups and spacing from a vector of IDs. If guessdups=TRUE, then getLayout calls getDupSpacing.

The function getLayout2 attempts to determine the print layout from the header information of an actual GAL file.

Value

A printlayout object, which is a list with the following components. The last two components are present only if guessdups=TRUE.

Author(s)

Gordon Smyth and James Wettenhall

See Also

An overview of LIMMA functions for reading data is given in 03.ReadingData.

Examples

# gal <- readGAL()
# layout <- getLayout(gal)

Description

Convert character to numerical spacing measure for within-array replicate spots.

Usage

getSpacing(spacing, layout)

Arguments

Details

"rows" means that duplicate spots are printed side-by-side by rows. These will be recorded in consecutive rows in the data object.