Package 'edgeR'

Description

edgeR is a package for the analysis of digital gene expression data arising from RNA sequencing technologies such as SAGE, CAGE, Tag-seq or RNA-seq, with emphasis on testing for differential expression. It can also be used for other sequencing technologies from which read counts are produced, such as ChIP-seq, Hi-C or CRISPR.

Particular strengths of the package include the ability to estimate biological variation between replicate libraries, and to conduct exact tests of significance which are suitable for small counts. The package is able to make use of even minimal numbers of replicates.

The supplied counts are assumed to be those of genes in a RNA-seq experiment. However, counts can be supplied for any genomic feature of interest, e.g., tags, transcripts, exons, or even arbitrary intervals of the genome.

An extensive User's Guide is available, and can be opened by typing edgeRUsersGuide() at the R prompt. Detailed help pages are also provided for each individual function.

The edgeR package implements original statistical methodology described in the publications below.

Author(s)

Yunshun Chen, Aaron TL Lun, Davis J McCarthy, Lizhong Chen, Pedro Baldoni, Matthew E Ritchie, Belinda Phipson, Yifang Hu, Xiaobei Zhou, Mark D Robinson, Gordon K Smyth

References

Baldoni PL, Chen L, Smyth GK (2024). Faster and more accurate assessment of differential transcript expression with Gibbs sampling and edgeR v4. NAR Genomics and Bioinformatics. doi:10.1101/2024.06.25.600555.

Chen Y, Chen L, Lun ATL, Baldoni PL, Smyth GK (2024). edgeR 4.0: powerful differential analysis of sequencing data with expanded functionality and improved support for small counts and larger datasets. bioRxiv 2024/576131. doi:10.1101/2024.01.21.576131

Baldoni PL#, Chen Y#, Hediyeh-zadeh S, Liao Y, Dong X, Ritchie ME, Shi W, Smyth GK (2024). Dividing out quantification uncertainty allows efficient assessment of differential transcript expression with edgeR. Nucleic Acids Research 52, e13.

Chen Y, Pal B, Visvader JE, Smyth GK (2017). Differential methylation analysis of reduced representation bisulfite sequencing experiments using edgeR. F1000Research 6, 2055.

Lun, AT, Smyth GK (2017). No counts, no variance: allowing for loss of degrees of freedom when assessing biological variability from RNA-seq data. Statistical Applications in Genetics and Molecular Biology 16(2), 83-93.

Chen Y, Lun ATL, Smyth GK (2016). From reads to genes to pathways: differential expression analysis of RNA-Seq experiments using Rsubread and the edgeR quasi-likelihood pipeline. F1000Research 5, 1438.

Lun ATL, Chen Y, Smyth GK (2016). It's DE-licious: a recipe for differential expression analyses of RNA-seq experiments using quasi-likelihood methods in edgeR. Methods in Molecular Biology 1418, 391-416. https://gksmyth.github.io/pubs/QLedgeRPreprint.pdf

Dai Z, Sheridan JM, Gearing LJ, Moore DL, Su S, Wormald S, Wilcox S, O'Connor L, Dickins RA, Blewitt ME, Ritchie ME (2014). edgeR: a versatile tool for the analysis of shRNA-seq and CRISPR-Cas9 genetic screens. F1000Research 3, 95.

Chen Y, Lun ATL, Smyth GK (2014). Differential expression analysis of complex RNA-seq experiments using edgeR. In: Statistical Analysis of Next Generation Sequence Data, Somnath Datta and Daniel S Nettleton (eds), Springer, New York, pages 51-74. https://gksmyth.github.io/pubs/edgeRChapterPreprint.pdf

Zhou X, Lindsay H, Robinson MD (2014). Robustly detecting differential expression in RNA sequencing data using observation weights. Nucleic Acids Research 42, e91.

Anders S, McCarthy DJ, Chen Y, Okoniewski M, Smyth GK, Huber W, Robinson MD (2013). Count-based differential expression analysis of RNA sequencing data using R and Bioconductor. Nature Protocols 8, 1765-1786.

McCarthy DJ#, Chen Y#, Smyth GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297.

Robinson MD, Oshlack A (2010). A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biology, 11, R25.

Robinson MD, McCarthy DJ, Smyth GK (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140

Robinson MD, Smyth GK (2008). Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics, 9, 321-332

Robinson MD, Smyth GK (2007). Moderated statistical tests for assessing differences in tag abundance. Bioinformatics 23, 2881-2887 )

Description

Add a library size-adjusted prior count to each observation.

Usage

addPriorCount(y, lib.size=NULL, offset=NULL, prior.count=1)

Arguments

Details

This function adds a positive prior count to each observation, often useful for avoiding zeroes during calculation of log-values. For example, predFC will call this function to calculate shrunken log-fold changes. aveLogCPM and cpm also use the same underlying code to calculate (average) log-counts per million.

The actual value added to the counts for each library is scaled according to the library size. This ensures that the relative contribution of the prior is the same for each library. Otherwise, a fixed prior would have little effect on a large library, but a big effect for a small library.

The library sizes are also modified, with twice the scaled prior being added to the library size for each library. To understand the motivation for this, consider that each observation is, effectively, a proportion of the total count in the library. The addition scheme implemented here represents an empirical logistic transform and ensures that the proportion can never be zero or one.

If offset is supplied, this is used in favour of lib.size where exp(offset) is defined as the vector/matrix of library sizes. If an offset matrix is supplied, this will lead to gene-specific scaling of the prior as described above.

Most use cases of this function will involve supplying a constant value to prior.count for all genes. However, it is also possible to use gene-specific values by supplying a vector of length equal to the number of rows in y.

Value

A list is returned containing y, a matrix of counts with the added priors; and offset, a CompressedMatrix containing the (log-transformed) modified library sizes.

Author(s)

Aaron Lun

See Also

aveLogCPM, cpm, predFC

Examples

original <- matrix(rnbinom(1000, mu=20, size=10), nrow=200)
head(original)

out <- addPriorCount(original)
head(out$y)
head(out$offset)

Description

Compute adjusted profile log-likelihoods for the dispersion parameters of genewise negative binomial glms.

Usage

adjustedProfileLik(dispersion, y, design, offset, weights=NULL, adjust=TRUE, 
            start=NULL, get.coef=FALSE)

Arguments

Details

For each row of data, compute the adjusted profile log-likelihood for the dispersion parameter of the negative binomial glm. The adjusted profile likelihood is described by McCarthy et al (2012) and is based on the method of Cox and Reid (1987).

The adjusted profile likelihood is an approximation to the log-likelihood function, conditional on the estimated values of the coefficients in the NB log-linear models. The conditional likelihood approach is a technique for adjusting the likelihood function to allow for the fact that nuisance parameters have to be estimated in order to evaluate the likelihood. When estimating the dispersion, the nuisance parameters are the coefficients in the log-linear model.

This implementation calls the LAPACK library to perform the Cholesky decomposition during adjustment estimation.

The purpose of start and get.coef is to allow hot-starting for multiple calls to adjustedProfileLik, when only the dispersion is altered. Specifically, the returned GLM coefficients from one call with get.coef==TRUE can be used as the start values for the next call.

The weights argument is interpreted in terms of averages. Each value of y is assumed to be the average of n independent and identically distributed NB counts, where n is given by the weight. This assumption can generalized to fractional weights.

Value

If get.coef==FALSE, a vector of adjusted profile log-likelihood values is returned containing one element for each row of y.

Otherwise, a list is returned containing apl, the aforementioned vector of adjusted profile likelihoods, and beta, the numeric matrix of fitted GLM coefficients.

Author(s)

Yunshun Chen, Gordon Smyth, Aaron Lun

References

Cox, DR, and Reid, N (1987). Parameter orthogonality and approximate conditional inference. Journal of the Royal Statistical Society Series B 49, 1-39.

McCarthy, DJ, Chen, Y, Smyth, GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297. doi:10.1093/nar/gks042

See Also

glmFit

Examples

y <- matrix(rnbinom(30, mu=10, size=20), 10, 3)
design <- matrix(1, 3, 1)
dispersion <- 0.05
adjustedProfileLik(dispersion, y, design, offset=0)

Description

Turn a TopTags object into a data.frame.

Usage

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

Arguments

Details

Convert edgeR objects into data.frames. This method returns the table component of a TopTags object. For DGEExact and DGELRT objects, the genes and table components are combined into a data.frame, similar to what is done by topTags but without sorting or p-value adjustment. For DGEList, the genes and counts components are combined into a data.frame.

Amongst other things, this functionality allows edgeR objects to be written to files using write.table or write.csv.

Value

A data.frame.

Author(s)

Gordon Smyth

See Also

as.data.frame in the base package.

Description

Coerce a digital gene expression object into a numeric matrix by extracting the count values.

Usage

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

Arguments

Details

This method extracts the matrix of counts from a DGEList or the matrix of coefficients from a DGEGLM fit.

This involves 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 as.matrix in the limma package.

Description

Compute average log2 counts-per-million for each row of counts.

Usage

## S3 method for class 'DGEList'
aveLogCPM(y, normalized.lib.sizes=TRUE, prior.count=2, dispersion=NULL, ...)
## S3 method for class 'SummarizedExperiment'
aveLogCPM(y, normalized.lib.sizes=TRUE, prior.count=2, 
          dispersion=NULL, ...)
## Default S3 method:
aveLogCPM(y, lib.size=NULL, offset=NULL, prior.count=2, dispersion=NULL,
          weights=NULL, ...)

Arguments

Details

This function uses mglmOneGroup to compute average counts-per-million (AveCPM) for each row of counts, and returns log2(AveCPM). An average value of prior.count is added to the counts before running mglmOneGroup. If prior.count is a vector, each entry will be added to all counts in the corresponding row of y, as described in addPriorCount.

This function is similar to

log2(rowMeans(cpm(y, ...))),

but with the refinement that larger library sizes are given more weight in the average. The two versions will agree for large values of the dispersion.

Value

Numeric vector giving log2(AveCPM) for each row of y.

Author(s)

Gordon Smyth

See Also

See cpm for individual logCPM values, rather than genewise averages.

Addition of the prior count is performed using the strategy described in addPriorCount.

The computations for aveLogCPM are done by mglmOneGroup.

Examples

y <- matrix(c(0,100,30,40),2,2)
lib.size <- c(1000,10000)

# With disp large, the function is equivalent to row-wise averages of individual cpms:
aveLogCPM(y, dispersion=1e4)
cpm(y, log=TRUE, prior.count=2)

# With disp=0, the function is equivalent to pooling the counts before dividing by lib.size:
aveLogCPM(y,prior.count=0,dispersion=0)
cpms <- rowSums(y)/sum(lib.size)*1e6
log2(cpms)

# The function works perfectly with prior.count or dispersion vectors:
aveLogCPM(y, prior.count=runif(nrow(y), 1, 5))
aveLogCPM(y, dispersion=runif(nrow(y), 0, 0.2))

Description

Computes p-values for differential abundance for each gene between two digital libraries, conditioning on the total count for each gene. The counts in each group as a proportion of the whole are assumed to follow a binomial distribution.

Usage

binomTest(y1, y2, n1=sum(y1), n2=sum(y2), p=n1/(n1+n2))

Arguments

Details

This function can be used to compare two libraries from SAGE, RNA-Seq, ChIP-Seq or other sequencing technologies with respect to technical variation.

An exact two-sided binomial test is computed for each gene. This test is closely related to Fisher's exact test for 2x2 contingency tables but, unlike Fisher's test, it conditions on the total number of counts for each gene. The null hypothesis is that the expected counts are in the same proportions as the library sizes, i.e., that the binomial probability for the first library is n1/(n1+n2).

The two-sided rejection region is chosen analogously to Fisher's test. Specifically, the rejection region consists of those values with smallest probabilities under the null hypothesis.

When the counts are reasonably large, the binomial test, Fisher's test and Pearson's chisquare all give the same results. When the counts are smaller, the binomial test is usually to be preferred in this context.

This function replaces the earlier sage.test functions in the statmod and sagenhaft packages. It produces the same results as binom.test in the stats packge, but is much faster.

Value

Numeric vector of p-values.

Author(s)

Gordon Smyth

References

https://en.wikipedia.org/wiki/Binomial_test

https://en.wikipedia.org/wiki/Fisher's_exact_test

https://en.wikipedia.org/wiki/Serial_analysis_of_gene_expression

https://en.wikipedia.org/wiki/RNA-Seq

See Also

sage.test (statmod package), binom.test (stats package)

Examples

binomTest(c(0,5,10),c(0,30,50),n1=10000,n2=15000)
#  Univariate equivalents:
binom.test(5,5+30,p=10000/(10000+15000))$p.value
binom.test(10,10+50,p=10000/(10000+15000))$p.value

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

## S3 method for class 'DGEList'
camera(y, index, design = NULL, contrast = ncol(design), weights = NULL,
       use.ranks = FALSE, allow.neg.cor=FALSE, inter.gene.cor=0.01, sort = TRUE, ...)
## S3 method for class 'DGEGLM'
camera(y, index, design = NULL, contrast = ncol(design), weights = NULL,
       use.ranks = FALSE, allow.neg.cor=FALSE, inter.gene.cor=0.01, sort = TRUE, ...)
## S3 method for class 'DGELRT'
cameraPR(statistic, index, use.ranks = FALSE, inter.gene.cor=0.01, sort = TRUE, ...)

Arguments

Details

Camera is a competitive gene set test proposed by Wu and Smyth (2012) for microarray data and camera and implemented as an S3 generic function in the limma package. It is often used for gene set enrichment analyses (GSEA) together with a database of gene sets such as the MSigDB. Here we provide camera methods for DGEList and DGEGLM class objects. The negative binomial count data is converted to approximate normal deviates by computing mid-p quantile residuals (Dunn and Smyth, 1996; Routledge, 1994), under the null hypothesis that the contrast is zero, and the normal deviates are then passed to limma's camera function.

The cameraPR function is a variation of the camera method for pre-ranked diffential expression statistics. We provide here a cameraPR method for DGELRT objects.

Value

A data.frame giving the gene set results, with a row for each gene set defined by index. If sort=TRUE then results are sorted in decreasing order of significance. See camera for details.

Author(s)

Yunshun Chen, Gordon Smyth

References

Dunn PK, Smyth GK (1996). Randomized quantile residuals. Journal of Computational and Graphical Statistics 5, 236-244. doi:10.1080/10618600.1996.10474708 https://gksmyth.github.io/pubs/residual.html

Routledge RD (1994). Practicing safe statistics with the mid-p. Canadian Journal of Statistics 22, 103-110.

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

See Also

camera.

Examples

mu <- matrix(10, 100, 4)
group <- factor(c(0,0,1,1))
design <- model.matrix(~group)

# First set of 10 genes that are genuinely differentially expressed
iset1 <- 1:10
mu[iset1,3:4] <- mu[iset1,3:4]+10

# Second set of 10 genes are not DE
iset2 <- 11:20

# Generate counts and create a DGEList object
y <- matrix(rnbinom(100*4, mu=mu, size=10),100,4)
y <- DGEList(counts=y, group=group)

# Estimate dispersions
y <- estimateDisp(y, design)

# Gene set tests
camera(y, iset1, design)
camera(y, iset2, design)
camera(y, list(set1=iset1,set2=iset2), design)

# Alternative pre-ranked version
fit <- glmQLFit(y, design)
q <- glmQLFTest(fit)
cameraPR(q, list(set1=iset1,set2=iset2))

Description

Read transcript counts from RSEM, kallisto or Salmon output for a series of biological samples and use bootstrap or posterior distribution samples to estimate the read-to-transcript-ambiguity for each transcript.

Usage

catchSalmon(paths, verbose = TRUE)
catchKallisto(paths, verbose = TRUE)
catchRSEM(files = NULL, ngibbs = 100, verbose = TRUE)

Arguments

Details

catchSalmon assumes that Salmon (Patro et al 2017; Zakeri et al 2017) has been run to estimate transcript counts for one or more RNA samples and that either bootstrap resamples or Gibbs posterior samples are included in the Salmon output. catchSalmon will automatically detect whether bootstrap or Gibbs samples are available. The number of technical resamples does not need to be the same for each RNA sample, but the resamples should always be of the same type, either bootstrap or Gibbs. We recommend that at least 200 bootstrap or Gibbs resamples are generated in total across all the RNA samples (Baldoni et al 2024b).

catchKallisto assumes that kallisto (Bray et al 2016) has been run to estimate transcript counts for one or more RNA samples and that bootstrap samples have also been generated. The number of bootstrap resamples does not need to be the same for each RNA sample.

catchRSEM reads *.isoforms.results files created by RSEM (Li and Dewey, 2011), which should contain means and standard deviations from Gibbs posterior samples as well as the estimated transcript counts. catchRSEM cannot detect the number of Gibbs samples performed for each RNA sample, so this must be specified via the ngibbs argument. The number of Gibbs samples is assumed to be the same for each RNA sample.

These functions read the transcript counts and use the technical (bootstrap or Gibbs) resamples to estimate an overdispersion parameter for each transcript. The overdispersion represents the variance inflation that occurs from ambiguity in assigning sequence reads to transcripts, a phenomenon that is called read to transcript ambiguity (RTA) overdispersion by Baldoni et al (2024ab). Transcripts that overlap other transcripts and have greater quantification uncertainty will have larger RTA overdispersions. The RTA overdispersions are greater than or equal to 1, with 1 representing no variance inflation.

To assess differential transcript expression, the transcript counts can be divided by the overdisperson parameters, after which the divided-counts can be input into standard differential expression pipelines designed for gene-level counts (Baldoni et al 2024a). The edgeR quasi pipeline has been found to perform well with the divided-counts (Baldoni et al 2024ab). The divided-counts behave much like negative binomial counts and show the same mean-variance trends as for gene-level RNA-seq counts. While bootstrap and Gibbs resamples both perform well, Baldoni et al (2024b) show that Gibbs resamples are computationally faster than bootstrap resamples and give slightly better performance in the downstream differential expression analyses.

Value

A list containing components

Author(s)

Gordon Smyth and Pedro Baldoni

References

Baldoni PL, Chen Y, Hediyeh-zadeh S, Liao Y, Dong X, Ritchie ME, Shi W, Smyth GK (2024a). Dividing out quantification uncertainty allows efficient assessment of differential transcript expression with edgeR. Nucleic Acids Research 52(3), e13. doi:10.1093/nar/gkad1167.

Baldoni PL, Chen L, Smyth GK (2024b). Faster and more accurate assessment of differential transcript expression with Gibbs sampling and edgeR v4. NAR Genomics and Bioinformatics 6(4), lqae151. doi:10.1093/nargab/lqae151.

Bray NL, Pimentel H, Melsted P, Pachter L (2016). Near-optimal probabilistic RNA-seq quantification. Nature Biotechnology, 34(5), 525-527.

Li B, Dewey CN (2011). RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC bioinformatics, 12, 323.

Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C (2017). Salmon provides fast and bias-aware quantification of transcript expression. Nature Methods, 14(4), 417-419.

Zakeri M, Srivastava A, Almodaresi F, Patro R (2017). Improved data-driven likelihood factorizations for transcript abundance estimation. Bioinformatics 33(14), i142-i151.

Examples

## Not run: 
# Read Salmon ouput and estimate overdispersion for each transcript
s <- catchSalmon(paths)

# Divide the transcript counts by the overdispersions ready for a standard edgeR DE analysis
dge <- DGEList(counts=s$counts/s$annotation$Overdispersion, genes=s$annotation)

## End(Not run)

Description

Combine a set of DGEList objects.

Usage

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

Arguments

Details

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

For cbind, the matrices of count data from the individual objects are cbinded. The data.frames of samples 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 count data are rbinded while the sample information is unchanged.

Value

An DGEList object holding data from all samples and all genes from the individual objects.

Author(s)

Gordon Smyth

See Also

cbind in the base package.

Examples

## Not run: 
dge <- cbind(dge1,dge2,dge3)

## End(Not run)

Description

Common conditional log-likelihood parameterized in terms of delta (phi / (phi+1))

Usage

commonCondLogLikDerDelta(y, delta, der = 0)

Arguments

Details

The common conditional log-likelihood is constructed by summing over all of the individual genewise conditional log-likelihoods. The common conditional log-likelihood is taken as a function of the dispersion parameter (phi), and here parameterized in terms of delta (phi / (phi+1)). The value of delta that maximizes the common conditional log-likelihood is converted back to the phi scale, and this value is the estimate of the common dispersion parameter used by all genes.

Value

numeric scalar of function/derivative evaluated at given delta

Author(s)

Davis McCarthy

See Also

estimateCommonDisp is the user-level function for estimating the common dispersion parameter.

Examples

counts<-matrix(rnbinom(20,size=1,mu=10),nrow=5)
d<-DGEList(counts=counts,group=rep(1:2,each=2),lib.size=rep(c(1000:1001),2))
y<-splitIntoGroups(d)
ll1<-commonCondLogLikDerDelta(y,delta=0.5,der=0)
ll2<-commonCondLogLikDerDelta(y,delta=0.5,der=1)

Description

Derivatives of the negative-binomial log-likelihood with respect to the dispersion parameter for each gene, conditional on the mean count, for a single group of replicate libraries of the same size.

Usage

condLogLikDerSize(y, r, der=1L)
condLogLikDerDelta(y, delta, der=1L)

Arguments

Details

The library sizes must be equalized before running this function. This function carries out the actual mathematical computations for the conditional log-likelihood and its derivatives, calculating the conditional log-likelihood for each gene. Derivatives are with respect to either the size (r) or the delta parametrization (delta) of the dispersion.

Value

vector of row-wise derivatives with respect to r or delta

Author(s)

Mark Robinson, Davis McCarthy, Gordon Smyth

Examples

y <- matrix(rnbinom(10,size=1,mu=10),nrow=5)
condLogLikDerSize(y,r=1,der=1)
condLogLikDerDelta(y,delta=0.5,der=1)

Description

Compute counts per million (CPM) or reads per kilobase per million (RPKM).

Usage

## S3 method for class 'DGEList'
cpm(y, normalized.lib.sizes = TRUE,
       log = FALSE, prior.count = 2, ...)
## S3 method for class 'SummarizedExperiment'
cpm(y, normalized.lib.sizes = TRUE,
       log = FALSE, prior.count = 2, ...)
## S3 method for class 'DGEGLM'
cpm(y, log = FALSE, shrunk = TRUE, ...)
## Default S3 method:
cpm(y, lib.size = NULL, offset=NULL,
       log = FALSE, prior.count = 2, ...)
## S3 method for class 'DGEList'
rpkm(y, gene.length = NULL, normalized.lib.sizes = TRUE,
       log = FALSE, prior.count = 2, ...)
## S3 method for class 'SummarizedExperiment'
rpkm(y, gene.length = NULL, normalized.lib.sizes = TRUE,
       log = FALSE, prior.count = 2, ...)
## S3 method for class 'DGEGLM'
rpkm(y, gene.length, log = FALSE, shrunk = TRUE, ...)
## Default S3 method:
rpkm(y, gene.length, lib.size = NULL, offset=NULL,
       log = FALSE, prior.count = 2, ...)
## S3 method for class 'DGEList'
cpmByGroup(y, group = NULL, dispersion = NULL, ...)
## S3 method for class 'SummarizedExperiment'
cpmByGroup(y, group = NULL, dispersion = NULL, ...)
## Default S3 method:
cpmByGroup(y, group = NULL, dispersion = 0.05,
       offset = NULL, weights = NULL, log = FALSE, prior.count = 2, ...)
## S3 method for class 'DGEList'
rpkmByGroup(y, group = NULL, gene.length = NULL, dispersion = NULL, ...)
## S3 method for class 'SummarizedExperiment'
rpkmByGroup(y, group = NULL, gene.length = NULL, dispersion = NULL, ...)
## Default S3 method:
rpkmByGroup(y, group = NULL, gene.length, dispersion = 0.05,
       offset = NULL, weights = NULL, log = FALSE, prior.count = 2, ...)

Arguments

Details

CPM or RPKM values are useful descriptive measures for the expression level of a gene. By default, the normalized library sizes are used in the computation for DGEList objects but simple column sums for matrices.

If log-values are computed, then a small count, given by prior.count but scaled to be proportional to the library size, is added to y to avoid taking the log of zero.

The rpkm methods for DGEList, DGEGLM or DGELRT objects will try to find the gene lengths in a column of y$genes called Length or length. Failing that, it will look for any column name containing "length" in any capitalization.

The cpm and rpkm methods for DGEGLM and DGELRT fitted model objects return fitted CPM or RPKM values. If shrunk=TRUE, then the CPM or RPKM values will reflect the prior.count input to the original linear model fit. If shrunk=FALSE, then the CPM or RPKM values will be computed with prior.count=0. Note that the latter could result in taking the log of near-zero values if log=TRUE.

cpmByGroup and rpkmByGroup compute group average values on the unlogged scale.

Value

A numeric matrix of CPM or RPKM values, on the log2 scale if log=TRUE. cpm and rpkm produce matrices of the same size as y. If y was a data object, then observed values are returned. If y was a fitted model object, then fitted values are returned.

cpmByGroup and rpkmByGroup produce matrices with a column for each level of group.

Note

aveLogCPM(y), rowMeans(cpm(y,log=TRUE)) and log2(rowMeans(cpm(y)) all give slightly different results.

Author(s)

Davis McCarthy, Gordon Smyth, Yunshun Chen, Aaron Lun

See Also

aveLogCPM

Examples

y <- matrix(rnbinom(20,size=1,mu=10),5,4)
cpm(y)

d <- DGEList(counts=y, lib.size=1001:1004)
cpm(d)
cpm(d,log=TRUE)

d$genes <- data.frame(Length=c(1000,2000,500,1500,3000))
rpkm(d)

cpmByGroup(d, group=c(1,1,2,2))

rpkmByGroup(d, group=c(1,1,2,2))

Description

Discretizes a numeric vector. Divides the range of x into intervals, so that each interval contains a minimum number of values, and codes the values in x according to which interval they fall into.

Usage

cutWithMinN(x, intervals=2, min.n=1)

Arguments

Details

This function strikes a compromise between the base functions cut, which by default cuts a vector into equal length intervals, and quantile, which is suited to finding equally populated intervals. It finds a partition of the x values that is as close as possible to equal length intervals while keeping at least min.n values in each interval.

Tied values of x are broken by random jittering, so the partition may vary slightly from run to run if there are many tied values.

Value

A list with components:

Author(s)

Gordon Smyth

See Also

cut, quantile.

Examples

x <- c(1,2,3,4,5,6,7,100)
cutWithMinN(x,intervals=3,min.n=1)

Description

Identify which genes are significantly differentially expressed from an edgeR test object containing p-values and test statistics.

Usage

## S3 method for class 'DGELRT'
decideTests(object, adjust.method="BH", p.value=0.05, lfc=0, ...)

Arguments

Details

This function applies a multiple testing procedure and significance level cutoff to the genewise tests contained in an edgeR test object and collates the results in a data.frame table.

The function can apply optionally apply a logFC cutoff and well as a p-value or FDR cutoff (although logFCs cutoff are not recommended, see note below). If the statistical tests are on 1 degree of freedom, then the logFC cutoff is applied to the absolute coefficient or contrast. If the statistical tests are on more than 1 degree of freedom, then the logFC cutoff will be satisfied if any of the coefficients or contrasts that define the test are greater than the cutoff.

Value

An object of class TestResults. This is essentially a single-column integer matrix with elements -1, 0 or 1 indicating whether each gene is classified as significantly down-regulated, not significant or significant up-regulated for the comparison contained in object. To be considered significant, genes need to have adjusted p-value below p.value and log2-fold-change greater than lfc.

If object contains F-tests or LRTs for multiple contrasts, then the genes are simply classified as significant (1) or not significant. In this case, the log2-fold-change theshold lfc has to be achieved by at least one of the contrasts for a gene to be significant.

Note

Although this function enables users to set p-value and logFC 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 also increase the family-wise error rate or false discovery rate above the nominal level. Users wanting to use fold change thresholding should considering using glmTreat instead and leaving lfc at the default value when using decideTests.

Author(s)

Davis McCarthy, Gordon Smyth and the edgeR team

See Also

decideTests and TestResults in the limma package.

Examples

ngenes <- 100
x1 <- rnorm(6)
x2 <- rnorm(6)
design <- cbind(Intercept=1,x1,x2)
beta <- matrix(0,ngenes,3)
beta[,1] <- 4
beta[1:20,2] <- rnorm(20)
mu <- 2^(beta %*% t(design))
y <- matrix(rnbinom(ngenes*6,mu=mu,size=10),ngenes,6)
fit <- glmFit(y,design,dispersion=0.1)
lrt <- glmLRT(fit,coef=2:3)
res <- decideTests(lrt,p.value=0.1)
summary(res)
lrt <- glmLRT(fit,coef=2)
res <- decideTests(lrt,p.value=0.1)
summary(res)

Description

A list-based S4 class for for storing results of a differential expression analysis for DGE data.

List Components

For objects of this class, rows correspond to genomic features and columns to statistics associated with the differential expression analysis. The genomic features are called genes, but in reality might correspond to transcripts, tags, exons etc.

Objects of this class contain the following list components:

table:

data frame containing columns for the log2-fold-change, logFC, the average log2-counts-per-million, logCPM, and the two-sided p-value PValue.

comparison:

vector giving the two experimental groups/conditions being compared.

genes:

a data frame containing information about each gene (can be NULL).

Methods

This class inherits directly from class list, so DGEExact objects can be manipulated as if they were ordinary lists. However they can also be treated as if they were matrices for the purposes of subsetting.

The dimensions, row names and column names of a DGEExact object are defined by those of table, see dim.DGEExact or dimnames.DGEExact.

DGEExact objects can be subsetted, see subsetting.

DGEExact objects also have a show method so that printing produces a compact summary of their contents.

Author(s)

edgeR team. First created by Mark Robinson and Davis McCarthy

See Also

Other classes defined in edgeR are DGEList-class, DGEGLM-class, DGELRT-class, TopTags-class

Description

A list-based S4 class for storing results of a GLM fit to each gene in a DGE dataset.

List Components

For objects of this class, rows correspond to genomic features and columns to coefficients in the linear model. The genomic features are called gene, but in reality might correspond to transcripts, tags, exons, etc.

Objects of this class contain the following list components:

coefficients:

matrix containing the coefficients computed from fitting the model defined by the design matrix to each gene in the dataset. Coefficients are on the natural log scale.

df.residual:

vector containing the residual degrees of freedom for the model fit to each gene in the dataset.

deviance:

vector giving the deviance from the model fit to each gene.

design:

design matrix for the full model from the likelihood ratio test.

offset:

scalar, vector or matrix of offset values to be included in the GLMs for each gene.

samples:

data frame containing information about the samples comprising the dataset.

genes:

data frame containing information about the tags for which we have DGE data (can be NULL if there is no information available).

dispersion:

scalar or vector providing the value of the dispersion parameter used in the negative binomial GLM for each gene.

lib.size:

vector providing the effective library size for each sample in the dataset.

weights:

matrix of weights used in the GLM fitting for each gene.

fitted.values:

the fitted (expected) values from the GLM for each gene.

AveLogCPM:

numeric vector giving average log2 counts per million for each gene.

Methods

This class inherits directly from class list so any operation appropriate for lists will work on objects of this class.

The dimensions, row names and column names of a DGEGLM object are defined by those of the dataset, see dim.DGEGLM or dimnames.DGEGLM.

DGEGLM objects can be subsetted, see subsetting.

DGEGLM objects also have a show method so that printing produces a compact summary of their contents.

Author(s)

edgeR team. First created by Davis McCarthy.

See Also

Other classes defined in edgeR are DGEList-class, DGEExact-class, DGELRT-class, TopTags-class

Description

Assembles a DGEList object from its components, especially the table counts as a matrix or data.frame.

Usage

## Default S3 method:
DGEList(counts, lib.size = NULL, norm.factors = NULL,
    samples = NULL, group = NULL, genes = NULL,
    remove.zeros = FALSE, ...)
## S3 method for class 'data.frame'
DGEList(counts, lib.size = NULL, norm.factors = NULL,
    samples = NULL, group = NULL, genes = NULL,
    remove.zeros = FALSE, annotation.columns = NULL, ...)

Arguments

Details

Assembles a DGEList object from its components. The only compulsory argument is the table of counts.

Normally, counts is a numeric matrix of counts but a data.frame is also allowed. If the counts is a data.frame, then the columns of the data.frame containing gene IDs or other gene annotation can be specified by annotation.columns, and the other columns are assumed to contain sequence read counts. If annotation.columns is not specified, then the function will check for non-numeric columns of counts and will attempt to set the leading columns up to the last non-numeric column as annotation.

Value

A DGEList object.

Author(s)

edgeR team. Originally created by Mark Robinson.

See Also

DGEList-class

Examples

ngenes <- 100
nsamples <- 4
Counts <- matrix(rnbinom(ngenes*nsamples,mu=5,size=10),ngenes,nsamples)
rownames(Counts) <- 1:ngenes
colnames(Counts) <- paste0("S",1:4)
Group <- gl(2,2)
Genes <- data.frame(Symbol=paste0("Gene",1:ngenes))
y <- DGEList(counts=Counts, group=Group, genes=Genes)
dim(y)
colnames(y)
y$samples
show(y)

Description

A list-based S4 class for storing read counts and associated information from digital gene expression or sequencing technologies.

List Components

For objects of this class, rows correspond to genomic features and columns to samples. The genomic features are called genes, but in reality might correspond to transcripts, tags, exons etc. Objects of this class contain the following essential list components:

counts:

numeric matrix of read counts, one row for each gene and one column for each sample.

samples:

data.frame with a row for each sample and columns group, lib.size and norm.factors containing the group labels, library sizes and normalization factors. Other columns can be optionally added to give more detailed sample information.

Optional components include:

genes:

data.frame giving annotation information for each gene. Same number of rows as counts.

AveLogCPM:

numeric vector giving average log2 counts per million for each gene.

common.dispersion:

numeric scalar giving the overall dispersion estimate.

trended.dispersion:

numeric vector giving trended dispersion estimates for each gene.

tagwise.dispersion:

numeric vector giving tagwise dispersion estimates for each gene (note that ‘tag’ and ‘gene’ are synonymous here).

offset:

numeric matrix of same size as counts giving offsets for use in log-linear models.

Methods

This class inherits directly from class list, so DGEList objects can be manipulated as if they were ordinary lists. However they can also be treated as if they were matrices for the purposes of subsetting.

The dimensions, row names and column names of a DGEList object are defined by those of counts, see dim.DGEList or dimnames.DGEList.

DGEList objects can be subsetted, see subsetting.

DGEList objects also have a show method so that printing produces a compact summary of their contents.

Author(s)

edgeR team. First created by Mark Robinson.

See Also

DGEList constructs DGEList objects. Other classes defined in edgeR are DGEExact-class, DGEGLM-class, DGELRT-class, TopTags-class

Description

A list-based S4 class for storing results of a GLM-based differential expression analysis for DGE data.

List Components

For objects of this class, rows correspond to genomic features and columns to statistics associated with the differential expression analysis. The genomic features are called genes, but in reality might correspond to transcripts, tags, exons etc.

Objects of this class contain the following list components:

table:

data frame containing the log-concentration (i.e. expression level), the log-fold change in expression between the two groups/conditions and the exact p-value for differential expression, for each gene.

coefficients.full:

matrix containing the coefficients computed from fitting the full model (fit using glmFit and a given design matrix) to each gene in the dataset.

coefficients.null:

matrix containing the coefficients computed from fitting the null model to each gene in the dataset. The null model is the model to which the full model is compared, and is fit using glmFit and dropping selected column(s) (i.e. coefficient(s)) from the design matrix for the full model.

design:

design matrix for the full model from the likelihood ratio test.

...:

if the argument y to glmLRT (which produces the DGELRT object) was itself a DGEList object, then the DGELRT will contain all of the elements of y, except for the table of counts and the table of pseudocounts.

Methods

This class inherits directly from class list, so DGELRT objects can be manipulated as if they were ordinary lists. However they can also be treated as if they were matrices for the purposes of subsetting.

The dimensions, row names and column names of a DGELRT object are defined by those of table, see dim.DGELRT or dimnames.DGELRT.

DGELRT objects can be subsetted, see subsetting.

DGELRT objects also have a show method so that printing produces a compact summary of their contents.

Author(s)

edgeR team. First created by Davis McCarthy

See Also

Other classes defined in edgeR are DGEList-class, DGEExact-class, DGEGLM-class, TopTags-class

Description

Given a negative binomial generalized log-linear model fit at the exon level, test for differential exon usage between experimental conditions.

Usage

diffSpliceDGE(glmfit, coef=ncol(glmfit$design), contrast=NULL, geneid, exonid=NULL,
              prior.count=0.125, robust=NULL, verbose=TRUE)

Arguments

Details

This function tests for differential exon usage for each gene for a given coefficient of the generalized linear model.

Testing for differential exon usage is equivalent to testing whether the exons in each gene have the same log-fold-changes as the other exons in the same gene. At exon-level, the log-fold-change of each exon is compared to the log-fold-change of the entire gene which contains that exon. At gene-level, two different tests are provided. One is converting exon-level p-values to gene-level p-values by the Simes method. The other is using exon-level test statistics to conduct gene-level tests.

Value

diffSpliceDGE produces an object of class DGELRT containing the component design from glmfit plus the following new components:

Some components of the output depend on whether glmfit is produced by glmFit or glmQLFit. If glmfit is produced by glmFit, then the following components are returned in the output object:

If glmfit is produced by glmQLFit, then the following components are returned in the output object:

The information and testing results for both exons and genes are sorted by geneid and by exonid within gene.

Author(s)

Yunshun Chen, Lizhong Chen and Gordon Smyth

Examples

# Gene exon annotation
Gene <- paste("Gene", 1:100, sep="")
Gene <- rep(Gene, each=10)
Exon <- paste("Ex", 1:10, sep="")
Gene.Exon <- paste(Gene, Exon, sep=".")
genes <- data.frame(GeneID=Gene, Gene.Exon=Gene.Exon)

group <- factor(rep(1:2, each=3))
design <- model.matrix(~group)
mu <- matrix(100, nrow=1000, ncol=6)
# knock-out the first exon of Gene1 by 90%
mu[1,4:6] <- 10
# generate exon counts
counts <- matrix(rnbinom(6000,mu=mu,size=20),1000,6)

y <- DGEList(counts=counts, lib.size=rep(1e6,6), genes=genes)
gfit <- glmFit(y, design, dispersion=0.05)

ds <- diffSpliceDGE(gfit, geneid="GeneID")
topSpliceDGE(ds)
plotSpliceDGE(ds)

Description

Retrieve the number of rows (genes) and columns (libraries) for an DGEList, DGEExact or TopTags Object.

Usage

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

Arguments

Details

Digital gene expression data objects share many analogies with ordinary matrices in which the rows correspond to 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 (libraries).

Author(s)

Gordon Smyth, Davis McCarthy

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 digital gene expression data object.

Usage

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

Arguments

Details

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

Setting dimension names is currently only permitted for DGEList or DGEGLM objects.

A consequence of these methods is that rownames, colnames, rownames<- and colnames<- will also work as expected on any of the above object classes.

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.

Description

Estimate the abundance-dispersion trend by computing the common dispersion for bins of genes of similar AveLogCPM and then fitting a smooth curve.

Usage

dispBinTrend(y, design=NULL, offset=NULL, df = 5, span=0.3, min.n=400,
             method.bin="CoxReid", method.trend="spline", AveLogCPM=NULL,
             weights=NULL, ...)

Arguments

Details

Estimate a dispersion parameter for each of many negative binomial generalized linear models by computing the common dispersion for genes sorted into bins based on overall AveLogCPM. A regression natural cubic splines or a linear loess curve is used to smooth the trend and extrapolate a value to each gene.

If there are fewer than min.n rows of y with at least one positive count, then one bin is used. The number of bins is limited to 1000.

Value

list with the following components:

Author(s)

Davis McCarthy and Gordon Smyth

References

McCarthy, DJ, Chen, Y, Smyth, GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297. doi:10.1093/nar/gks042

See Also

estimateGLMTrendedDisp

Examples

ngenes <- 1000
nlibs <- 4
means <- seq(5,10000,length.out=ngenes)
y <- matrix(rnbinom(ngenes*nlibs,mu=rep(means,nlibs),size=0.1*means),nrow=ngenes,ncol=nlibs)
keep <- rowSums(y) > 0
y <- y[keep,]
group <- factor(c(1,1,2,2))
design <- model.matrix(~group) # Define the design matrix for the full model
out <- dispBinTrend(y, design, min.n=100, span=0.3)
with(out, plot(AveLogCPM, sqrt(dispersion)))

Description

Estimate a common dispersion parameter across multiple negative binomial generalized linear models.

Usage

dispCoxReid(y, design=NULL, offset=NULL, weights=NULL, AveLogCPM=NULL, interval=c(0,4),
            tol=1e-5, min.row.sum=5, subset=10000)
dispDeviance(y, design=NULL, offset=NULL, interval=c(0,4), tol=1e-5, min.row.sum=5,
            subset=10000, AveLogCPM=NULL, robust=FALSE, trace=FALSE)
dispPearson(y, design=NULL, offset=NULL, min.row.sum=5, subset=10000,
            AveLogCPM=NULL, tol=1e-6, trace=FALSE, initial.dispersion=0.1)

Arguments

Details

These are low-level (non-object-orientated) functions called by estimateGLMCommonDisp.

dispCoxReid maximizes the Cox-Reid adjusted profile likelihood (Cox and Reid, 1987). dispPearson sets the average Pearson goodness of fit statistics to its (asymptotic) expected value. This is also known as the pseudo-likelihood estimator. dispDeviance sets the average residual deviance statistic to its (asymptotic) expected values. This is also known as the quasi-likelihood estimator.

Robinson and Smyth (2008) and McCarthy et al (2011) showed that the Pearson (pseudo-likelihood) estimator typically under-estimates the true dispersion. It can be seriously biased when the number of libraries (ncol(y) is small. On the other hand, the deviance (quasi-likelihood) estimator typically over-estimates the true dispersion when the number of libraries is small. Robinson and Smyth (2008) and McCarthy et al (2011) showed the Cox-Reid estimator to be the least biased of the three options.

dispCoxReid uses optimize to maximize the adjusted profile likelihood. dispDeviance uses uniroot to solve the estimating equation. The robust options use an order statistic instead the mean statistic, and have the effect that a minority of genes with very large (outlier) dispersions should have limited influence on the estimated value. dispPearson uses a globally convergent Newton iteration.

Value

Numeric vector of length one giving the estimated common dispersion.

Author(s)

Gordon Smyth

References

Cox, DR, and Reid, N (1987). Parameter orthogonality and approximate conditional inference. Journal of the Royal Statistical Society Series B 49, 1-39.

Robinson MD and Smyth GK (2008). Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics, 9, 321-332

McCarthy, DJ, Chen, Y, Smyth, GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research. http://nar.oxfordjournals.org/content/early/2012/02/06/nar.gks042 (Published online 28 January 2012)

See Also

estimateGLMCommonDisp, optimize, uniroot

Examples

ngenes <- 100
nlibs <- 4
y <- matrix(rnbinom(ngenes*nlibs,mu=10,size=10),nrow=ngenes,ncol=nlibs)
group <- factor(c(1,1,2,2))
lib.size <- rowSums(y)
design <- model.matrix(~group)
disp <- dispCoxReid(y, design, offset=log(lib.size), subset=100)

Description

Estimate genewise dispersion parameters across multiple negative binomial generalized linear models using weighted Cox-Reid Adjusted Profile-likelihood and cubic spline interpolation over a genewise grid.

Usage

dispCoxReidInterpolateTagwise(y, design, offset=NULL, dispersion, trend=TRUE,
                              AveLogCPM=NULL, min.row.sum=5, prior.df=10,
                              span=0.3, grid.npts=11, grid.range=c(-6,6),
                              weights=NULL)

Arguments

Details

In the edgeR context, dispCoxReidInterpolateTagwise is a low-level function called by estimateGLMTagwiseDisp.

dispCoxReidInterpolateTagwise calls the function maximizeInterpolant to fit cubic spline interpolation over a genewise grid.

Note that the terms ‘tag’ and ‘gene’ are synonymous here. The function is only named ‘Tagwise’ for historical reasons.

Value

dispCoxReidInterpolateTagwise produces a vector of genewise dispersions having the same length as the number of genes in the count data.

Author(s)

Yunshun Chen, Gordon Smyth

References

Cox, DR, and Reid, N (1987). Parameter orthogonality and approximate conditional inference. Journal of the Royal Statistical Society Series B 49, 1-39.

McCarthy, DJ, Chen, Y, Smyth, GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297. doi:10.1093/nar/gks042

See Also

estimateGLMTagwiseDisp, maximizeInterpolant

Examples

y <- matrix(rnbinom(1000, mu=10, size=2), ncol=4)
design <- matrix(1, 4, 1)
dispersion <- 0.5
d <- dispCoxReidInterpolateTagwise(y, design, dispersion=dispersion)
d

Description

Estimate trended dispersion parameters across multiple negative binomial generalized linear models using Cox-Reid adjusted profile likelihood.

Usage

dispCoxReidSplineTrend(y, design, offset=NULL, df = 5, subset=10000, AveLogCPM=NULL,
                       method.optim="Nelder-Mead", trace=0)
dispCoxReidPowerTrend(y, design, offset=NULL, subset=10000, AveLogCPM=NULL,
                       method.optim="Nelder-Mead", trace=0)

Arguments

Details

In the edgeR context, these are low-level functions called by estimateGLMTrendedDisp.

dispCoxReidSplineTrend and dispCoxReidPowerTrend fit abundance trends to the genewise dispersions. dispCoxReidSplineTrend fits a regression spline whereas dispCoxReidPowerTrend fits a log-linear trend of the form a*exp(abundance)^b+c. In either case, optim is used to maximize the adjusted profile likelihood (Cox and Reid, 1987).

Value

List containing numeric vectors dispersion and abundance containing the estimated dispersion and abundance for each gene. The vectors are of the same length as nrow(y).

Author(s)

Yunshun Chen, Davis McCarthy, Gordon Smyth

References

Cox, DR, and Reid, N (1987). Parameter orthogonality and approximate conditional inference. Journal of the Royal Statistical Society Series B 49, 1-39.

See Also

estimateGLMTrendedDisp

Examples

design <- matrix(1,4,1)
y <- matrix((rnbinom(400,mu=100,size=5)),100,4)
d1 <- dispCoxReidSplineTrend(y, design, df=3)
d2 <- dispCoxReidPowerTrend(y, design)
with(d2,plot(AveLogCPM,sqrt(dispersion)))

Description

Reform a factor so that only necessary levels are kept.

Usage

dropEmptyLevels(x)

Arguments

Details

In general, the levels of a factor, levels(x), may include values that never actually occur. This function drops any levels of that do not occur.

If x is not a factor, then the function returns factor(x). If x is a factor, then the function returns the same value as factor(x) or x[,drop=TRUE] but somewhat more efficiently.

Value

A factor with the same values as x but with a possibly reduced set of levels.

Author(s)

Gordon Smyth

See Also

factor.

Examples

x <- factor(c("a","b"), levels=c("c","b","a"))
x
dropEmptyLevels(x)

Description

Finds the location of the edgeR User's Guide and optionally opens it.

Usage

edgeRUsersGuide(view=TRUE)

Arguments

Details

The function vignette("edgeR") will find the short edgeR Vignette which describes how to obtain the edgeR User's Guide. The User's Guide is not itself a true vignette because it is not automatically generated using Sweave during the package build process. This means that it cannot be found using vignette, hence the need for this special function.

If the operating system is other than Windows, then the PDF viewer used is that given by Sys.getenv("R_PDFVIEWER"). The PDF viewer can be changed using Sys.putenv(R_PDFVIEWER=).

Value

Character string giving the file location. If view=TRUE, the PDF document reader is started and the User's Guide is opened, as a side effect.

Author(s)

Gordon Smyth

See Also

system

Examples

# To get the location:
edgeRUsersGuide(view=FALSE)
# To open in pdf viewer:
## Not run: edgeRUsersGuide()

Description

Adjusts counts so that the effective library sizes are equal, preserving fold-changes between groups and preserving biological variability within each group.

Usage

## S3 method for class 'DGEList'
equalizeLibSizes(y, dispersion=NULL, ...)
## Default S3 method:
equalizeLibSizes(y, group=NULL, dispersion=NULL, 
            lib.size=NULL, ...)

Arguments

Details

Thus function implements the quantile-quantile normalization method of Robinson and Smyth (2008). It computes normalized counts, or pseudo-counts, used by exactTest and estimateCommonDisp.

The output pseudo-counts are the counts that would have theoretically arisen had the effective library sizes been equal for all samples. The pseudo-counts are computed in such as way as to preserve fold-change differences beween the groups defined by y$samples$group as well as biological variability within each group. Consequently, the results will depend on how the groups are defined.

Note that the column sums of the pseudo.counts matrix will not generally be equal, because the effective library sizes are not necessarily the same as actual library sizes and because the normalized pseudo counts are not equal to expected counts.

Value

equalizeLibSizes.default returns a list with components:

equalizeLibSizes.DGEList returns a DGEList object with the above two components added.

Note

This function is intended mainly for internal edgeR use. It is not normally called directly by users.

Author(s)

Mark Robinson, Davis McCarthy, Gordon Smyth

References

Robinson MD and Smyth GK (2008). Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics, 9, 321-332. http://biostatistics.oxfordjournals.org/content/9/2/321

See Also

q2qnbinom

Examples

ngenes <- 1000
nlibs <- 2
counts <- matrix(0,ngenes,nlibs)
colnames(counts) <- c("Sample1","Sample2")
counts[,1] <- rpois(ngenes,lambda=10)
counts[,2] <- rpois(ngenes,lambda=20)
summary(counts)
y <- DGEList(counts=counts)
out <- equalizeLibSizes(y)
summary(out$pseudo.counts)

Description

Maximizes the negative binomial conditional common likelihood to estimate a common dispersion value across all genes.

Usage

## S3 method for class 'DGEList'
estimateCommonDisp(y, tol=1e-06, rowsum.filter=5, verbose=FALSE, ...)
## Default S3 method:
estimateCommonDisp(y, group=NULL, lib.size=NULL, tol=1e-06, 
          rowsum.filter=5, verbose=FALSE, ...)

Arguments

Details

Implements the conditional maximum likelihood (CML) method proposed by Robinson and Smyth (2008) for estimating a common dispersion parameter. This method proves to be accurate and nearly unbiased even for small counts and small numbers of replicates.

The CML method involves computing a matrix of quantile-quantile normalized counts, called pseudo-counts. The pseudo-counts are adjusted in such a way that the library sizes are equal for all samples, while preserving differences between groups and variability within each group. The pseudo-counts are included in the output of the function, but are intended mainly for internal edgeR use.

Value

estimateCommonDisp.DGEList adds the following components to the input DGEList object:

estimateCommonDisp.default returns a numeric scalar of the common dispersion estimate.

Author(s)

Mark Robinson, Davis McCarthy, Gordon Smyth

References

Robinson MD and Smyth GK (2008). Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics, 9, 321-332. http://biostatistics.oxfordjournals.org/content/9/2/321

See Also

equalizeLibSizes, estimateTrendedDisp, estimateTagwiseDisp

Examples

# True dispersion is 1/5=0.2
y <- matrix(rnbinom(250*4,mu=20,size=5),nrow=250,ncol=4)
dge <- DGEList(counts=y,group=c(1,1,2,2))
dge <- estimateCommonDisp(dge, verbose=TRUE)

Description

Maximizes the negative binomial likelihood to give the estimate of the common, trended and tagwise dispersions across all tags.

Usage

## S3 method for class 'DGEList'
estimateDisp(y, design=NULL, prior.df=NULL, trend.method="locfit", tagwise=TRUE,
          span=NULL, min.row.sum=5, grid.length=21, grid.range=c(-10,10),
          robust=FALSE, winsor.tail.p=c(0.05,0.1), tol=1e-06, ...)
## S3 method for class 'SummarizedExperiment'
estimateDisp(y, design=NULL, prior.df=NULL, trend.method="locfit", tagwise=TRUE,
          span=NULL, min.row.sum=5, grid.length=21, grid.range=c(-10,10),
          robust=FALSE, winsor.tail.p=c(0.05,0.1), tol=1e-06, ...)
## Default S3 method:
estimateDisp(y, design=NULL, group=NULL, lib.size=NULL, offset=NULL,
          prior.df=NULL, trend.method="locfit", tagwise=TRUE, span=NULL,
          min.row.sum=5, grid.length=21, grid.range=c(-10,10),
          robust=FALSE, winsor.tail.p=c(0.05,0.1), tol=1e-06, weights=NULL, ...)

Arguments

Details

This function calculates a matrix of likelihoods for each tag at a set of dispersion grid points, and then applies weighted likelihood empirical Bayes method to obtain posterior dispersion estimates. If there is no design matrix, it calculates the quantile conditional likelihood for each tag and then maximizes it. In this case, it is similar to the function estimateCommonDisp and estimateTagwiseDisp. If a design matrix is given, it calculates the adjusted profile log-likelihood for each tag and then maximizes it. In this case, it is similar to the functions estimateGLMCommonDisp, estimateGLMTrendedDisp and estimateGLMTagwiseDisp.

Note that the terms ‘tag’ and ‘gene’ are synonymous here.

Value

estimateDisp.DGEList adds the following components to the input DGEList object:

estimateDisp.SummarizedExperiment converts the input SummarizedExperiment object into a DGEList object, and then calls estimateDisp.DGEList. The output is a DGEList object.

estimateDisp.default returns a list containing common.dispersion, trended.dispersion, tagwise.dispersion (if tagwise=TRUE), span, prior.df and prior.n.

Note

The estimateDisp function doesn't give exactly the same estimates as the traditional calling sequences.

Author(s)

Yunshun Chen, Gordon Smyth

References

Chen, Y, Lun, ATL, and Smyth, GK (2014). Differential expression analysis of complex RNA-seq experiments using edgeR. In: Statistical Analysis of Next Generation Sequence Data, Somnath Datta and Daniel S. Nettleton (eds), Springer, New York, pages 51-74. https://gksmyth.github.io/pubs/edgeRChapterPreprint.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

estimateCommonDisp, estimateTagwiseDisp, estimateGLMCommonDisp, estimateGLMTrendedDisp, estimateGLMTagwiseDisp

Examples

# True dispersion is 1/5=0.2
y <- matrix(rnbinom(1000, mu=10, size=5), ncol=4)
group <- factor(c(1,1,2,2))
design <- model.matrix(~group)
d <- DGEList(counts=y, group=group)
d1 <- estimateDisp(d)
d2 <- estimateDisp(d, design)

Description

Estimate a dispersion value for each gene from exon-level count data by collapsing exons into the genes to which they belong.

Usage

estimateExonGenewiseDisp(y, geneID, group=NULL)

Arguments

Details

This function can be used to compute genewise dispersion estimates (for an experiment with a one-way, or multiple group, layout) from exon-level count data. estimateCommonDisp and estimateTagwiseDisp are used to do the computation and estimation, and the default arguments for those functions are used.

Value

estimateExonGenewiseDisp returns a vector of genewise dispersion estimates, one for each unique geneID.

Author(s)

Davis McCarthy, Gordon Smyth

See Also

estimateCommonDisp and related functions for estimating the dispersion parameter for the negative binomial model.

Examples

# generate exon counts from NB, create list object
y<-matrix(rnbinom(40,size=1,mu=10),nrow=10)
d<-DGEList(counts=y,group=rep(1:2,each=2))
genes <- rep(c("gene.1","gene.2"), each=5)
estimateExonGenewiseDisp(d, genes)

Description

Estimates a common negative binomial dispersion parameter for a DGE dataset with a general experimental design.

Usage

## S3 method for class 'DGEList'
estimateGLMCommonDisp(y, design=NULL, method="CoxReid",
                      subset=10000, verbose=FALSE, ...)
## Default S3 method:
estimateGLMCommonDisp(y, design=NULL, offset=NULL,
                      method="CoxReid", subset=10000, AveLogCPM=NULL,
                      verbose=FALSE, weights=NULL,...)

Arguments

Details

This function calls dispCoxReid, dispPearson or dispDeviance depending on the method specified. See dispCoxReid for details of the three methods and a discussion of their relative performance.

Value

The default method returns a numeric vector of length 1 containing the estimated common dispersion.

The DGEList method returns the same DGEList y as input but with common.dispersion as an added component. The output object will also contain a component AveLogCPM if it was not already present in y.

Author(s)

Gordon Smyth, Davis McCarthy, Yunshun Chen

References

McCarthy, DJ, Chen, Y, Smyth, GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297. doi:10.1093/nar/gks042

See Also

dispCoxReid, dispPearson, dispDeviance

estimateGLMTrendedDisp for trended dispersions or estimateGLMTagwiseDisp for genewise dispersions in the context of a generalized linear model.

estimateCommonDisp for the common dispersion or estimateTagwiseDisp for genewise dispersions in the context of a multiple group experiment (one-way layout).

Examples

#  True dispersion is 1/size=0.1
y <- matrix(rnbinom(1000,mu=10,size=10),ncol=4)
d <- DGEList(counts=y,group=c(1,1,2,2))
design <- model.matrix(~group, data=d$samples)
d1 <- estimateGLMCommonDisp(d, design, verbose=TRUE)

#  Compare with classic CML estimator:
d2 <- estimateCommonDisp(d, verbose=TRUE)

#  See example(glmFit) for a different example

Description

Compute a robust estimate of the negative binomial dispersion parameter for each gene, with expression levels specified by a log-linear model, using observation weights. These observation weights will be stored and used later for estimating regression parameters.

Usage

estimateGLMRobustDisp(y, design = NULL, prior.df = 10, update.trend = TRUE,
                      trend.method = "bin.loess", maxit = 6, k = 1.345,
                      residual.type = "pearson", verbose = FALSE,
                      record = FALSE)

Arguments

Details

Moderation of dispersion estimates towards a trend can be sensitive to outliers, resulting in an increase in false positives. That is, since the dispersion estimates are moderated downwards toward the trend and because the regression parameter estimates may be affected by the outliers, some genes are incorrectly deemed to be significantly differentially expressed. This function uses an iterative procedure where weights are calculated from residuals and estimates are made after re-weighting.

The robustly computed genewise estimates are reported in the tagwise.dispersion vector of the returned DGEList. The terms ‘tag’ and ‘gene’ are synonymous in this context.

Note: it is not necessary to first calculate the common, trended and genewise dispersion estimates. If these are not available, the function will first calculate this (in an unweighted) fashion.

Value

estimateGLMRobustDisp produces a DGEList object, which contains the (robust) genewise dispersion parameter estimate for each gene for the negative binomial model that maximizes the weighted Cox-Reid adjusted profile likelihood, as well as the observation weights. The observation weights are calculated using residuals and the Huber function.

Note that when record=TRUE, a simple list of DGEList objects is returned, one for each iteration (this is for debugging or tracking purposes).

Author(s)

Xiaobei Zhou, Mark D. Robinson

References

Zhou X, Lindsay H, Robinson MD (2014). Robustly detecting differential expression in RNA sequencing data using observation weights. Nucleic Acids Research, 42(11), e91.

See Also

This function calls estimateGLMTrendedDisp and estimateGLMTagwiseDisp.

Examples

y <- matrix(rnbinom(100*6,mu=10,size=1/0.1),ncol=6)
d <- DGEList(counts=y,group=c(1,1,1,2,2,2),lib.size=c(1000:1005))
d <- normLibSizes(d)
design <- model.matrix(~group, data=d$samples) # Define the design matrix for the full model
d <- estimateGLMRobustDisp(d, design)
summary(d$tagwise.dispersion)

Description

Compute an empirical Bayes estimate of the negative binomial dispersion parameter for each tag, with expression levels specified by a log-linear model.

Usage

## S3 method for class 'DGEList'
estimateGLMTagwiseDisp(y, design=NULL, prior.df=10,
            trend=!is.null(y$trended.dispersion), span=NULL, ...)
## Default S3 method:
estimateGLMTagwiseDisp(y, design=NULL, offset=NULL, dispersion,
            prior.df=10, trend=TRUE, span=NULL, AveLogCPM=NULL,
            weights=NULL, ...)

Arguments

Details

This function implements the empirical Bayes strategy proposed by McCarthy et al (2012) for estimating the tagwise negative binomial dispersions. The experimental conditions are specified by design matrix allowing for multiple explanatory factors. The empirical Bayes posterior is implemented as a conditional likelihood with tag-specific weights, and the conditional likelihood is computed using Cox-Reid approximate conditional likelihood (Cox and Reid, 1987).

The prior degrees of freedom determines the weight given to the global dispersion trend. The larger the prior degrees of freedom, the more the tagwise dispersions are squeezed towards the global trend.

Note that the terms ‘tag’ and ‘gene’ are synonymous here. The function is only named ‘Tagwise’ for historical reasons.

This function calls the lower-level function dispCoxReidInterpolateTagwise.

Value

estimateGLMTagwiseDisp.DGEList produces a DGEList object, which contains the tagwise dispersion parameter estimate for each tag for the negative binomial model that maximizes the Cox-Reid adjusted profile likelihood. The tagwise dispersions are simply added to the DGEList object provided as the argument to the function.

estimateGLMTagwiseDisp.default returns a vector of the tagwise dispersion estimates.

Author(s)

Gordon Smyth, Davis McCarthy

References

Cox, DR, and Reid, N (1987). Parameter orthogonality and approximate conditional inference. Journal of the Royal Statistical Society Series B 49, 1-39.

McCarthy, DJ, Chen, Y, Smyth, GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297. doi:10.1093/nar/gks042

See Also

estimateGLMCommonDisp for common dispersion or estimateGLMTrendedDisp for trended dispersion in the context of a generalized linear model.

estimateCommonDisp for common dispersion or estimateTagwiseDisp for tagwise dispersions in the context of a multiple group experiment (one-way layout).

Examples

y <- matrix(rnbinom(1000,mu=10,size=10),ncol=4)
d <- DGEList(counts=y,group=c(1,1,2,2),lib.size=c(1000:1003))
design <- model.matrix(~group, data=d$samples) # Define the design matrix for the full model
d <- estimateGLMTrendedDisp(d, design, min.n=10)
d <- estimateGLMTagwiseDisp(d, design)
summary(d$tagwise.dispersion)

Description

Estimates the abundance-dispersion trend by Cox-Reid approximate profile likelihood.

Usage

## S3 method for class 'DGEList'
estimateGLMTrendedDisp(y, design=NULL, method="auto", ...)
## Default S3 method:
estimateGLMTrendedDisp(y, design=NULL, offset=NULL, AveLogCPM=NULL,
                       method="auto", weights=NULL, ...)

Arguments

Details

Estimates the dispersion parameter for each gene with a trend that depends on the overall level of expression for that gene. This is done for a DGE dataset for general experimental designs by using Cox-Reid approximate conditional inference for a negative binomial generalized linear model for each gene with the unadjusted counts and design matrix provided.

The function provides an object-orientated interface to lower-level functions.

Value

When the input object is a DGEList, estimateGLMTrendedDisp produces a DGEList object, which contains the estimates of the trended dispersion parameter for the negative binomial model according to the method applied.

When the input object is a numeric matrix, it returns a vector of trended dispersion estimates calculated by one of the lower-level functions dispBinTrend, dispCoxReidPowerTrend and dispCoxReidSplineTrend.

Author(s)

Gordon Smyth, Davis McCarthy, Yunshun Chen

References

Cox, DR, and Reid, N (1987). Parameter orthogonality and approximate conditional inference. Journal of the Royal Statistical Society Series B 49, 1-39.

McCarthy, DJ, Chen, Y, Smyth, GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297. doi:10.1093/nar/gks042

See Also

dispBinTrend, dispCoxReidPowerTrend and dispCoxReidSplineTrend for details on how the calculations are done.

Examples

ngenes <- 250
nlibs <- 4
y <- matrix(rnbinom(ngenes*nlibs,mu=10,size=10),ngenes,nlibs)
d <- DGEList(counts=y,group=c(1,1,2,2),lib.size=c(1000:1003))
design <- model.matrix(~group, data=d$samples)
disp <- estimateGLMTrendedDisp(d, design, min.n=25, df=3)
plotBCV(disp)

Description

Estimates tagwise dispersion values by an empirical Bayes method based on weighted conditional maximum likelihood.

Usage

## S3 method for class 'DGEList'
estimateTagwiseDisp(y, prior.df=10, trend="movingave", span=NULL, method="grid", 
           grid.length=11, grid.range=c(-6,6), tol=1e-06, verbose=FALSE, ...)
## Default S3 method:
estimateTagwiseDisp(y, group=NULL, lib.size=NULL, dispersion, AveLogCPM=NULL, 
           prior.df=10, trend="movingave", span=NULL, method="grid", grid.length=11, 
           grid.range=c(-6,6), tol=1e-06, verbose=FALSE, ...)

Arguments

Details

This function implements the empirical Bayes strategy proposed by Robinson and Smyth (2007) for estimating the tagwise negative binomial dispersions. The experimental design is assumed to be a oneway layout with one or more experimental groups. The empirical Bayes posterior is implemented as a conditional likelihood with tag-specific weights.

The prior values for the dispersions are determined by a global trend. The individual tagwise dispersions are then squeezed towards this trend. The prior degrees of freedom determines the weight given to the prior. The larger the prior degrees of freedom, the more the tagwise dispersions are squeezed towards the global trend. If the number of libraries is large, the prior becomes less important and the tagwise dispersion are determined more by the individual tagwise data.

If trend="none", then the prior dispersion is just a constant, the common dispersion. Otherwise, the trend is determined by a moving average (trend="movingave") or loess smoother applied to the tagwise conditional log-likelihood. method="loess" applies a loess curve of degree 0 as implemented in loessByCol.

method="optimize" is not recommended for routine use as it is very slow. It is included for testing purposes.

Note that the terms ‘tag’ and ‘gene’ are synonymous here. The function is only named ‘Tagwise’ for historical reasons.

Value

estimateTagwiseDisp.DGEList adds the following components to the input DGEList object:

estimateTagwiseDisp.default returns a numeric vector of the tagwise dispersion estimates.

Author(s)

Mark Robinson, Davis McCarthy, Yunshun Chen and Gordon Smyth

References

Robinson, MD, and Smyth, GK (2007). Moderated statistical tests for assessing differences in tag abundance. Bioinformatics 23, 2881-2887. doi:10.1093/bioinformatics/btm453

See Also

estimateCommonDisp is usually run before estimateTagwiseDisp.

movingAverageByCol and loessByCol implement the moving average or loess smoothers.

Examples

# True dispersion is 1/5=0.2
y <- matrix(rnbinom(250*4,mu=20,size=5),nrow=250,ncol=4)
dge <- DGEList(counts=y,group=c(1,1,2,2))
dge <- estimateCommonDisp(dge)
dge <- estimateTagwiseDisp(dge)

Description

Estimates trended dispersion values by an empirical Bayes method.

Usage

## S3 method for class 'DGEList'
estimateTrendedDisp(y, method="bin.spline", df=5, span=2/3, ...)
## Default S3 method:
estimateTrendedDisp(y, group=NULL, lib.size=NULL, AveLogCPM=NULL, 
            method="bin.spline", df=5, span=2/3, ...)

Arguments

Details

This function takes the binned common dispersion and abundance, and fits a smooth curve through these binned values using either natural cubic splines or loess. From this smooth curve it predicts the dispersion value for each gene based on the gene's overall abundance. This results in estimates for the NB dispersion parameter which have a dependence on the overall expression level of the gene, and thus have an abundance-dependent trend.

Value

An object of class DGEList with the same components as for estimateCommonDisp plus the trended dispersion estimates for each gene.

Author(s)

Yunshun Chen and Gordon Smyth

See Also

estimateCommonDisp estimates a common value for the dispersion parameter for all genes - should generally be run before estimateTrendedDisp.

Examples

ngenes <- 1000
nlib <- 4
log2cpm <- seq(from=0,to=16,length=ngenes)
lib.size <- 1e7
mu <- 2^log2cpm * lib.size * 1e-6
dispersion <- 1/sqrt(mu) + 0.1
counts <- rnbinom(ngenes*nlib, mu=mu, size=1/dispersion)
counts <- matrix(counts,ngenes,nlib)
y <- DGEList(counts,lib.size=rep(lib.size,nlib))
y <- estimateCommonDisp(y)
y <- estimateTrendedDisp(y)

Description

Compute genewise exact tests for differences in the means between two groups of negative-binomially distributed counts.

Usage

exactTest(object, pair=1:2, dispersion="auto", rejection.region="doubletail",
          big.count=900, prior.count=0.125)
exactTestDoubleTail(y1, y2, dispersion=0, big.count=900)
exactTestBySmallP(y1, y2, dispersion=0)
exactTestByDeviance(y1, y2, dispersion=0)
exactTestBetaApprox(y1, y2, dispersion=0)

Arguments

Details

The functions test for differential expression between two groups of count libraries. They implement the exact test proposed by Robinson and Smyth (2008) for a difference in mean between two groups of negative binomial random variables. The functions accept two groups of count libraries, and a test is performed for each row of data. For each row, the test is conditional on the sum of counts for that row. The test can be viewed as a generalization of the well-known exact binomial test (implemented in binomTest) but generalized to overdispersed counts.

exactTest is the main user-level function, and produces an object containing all the necessary components for downstream analysis. exactTest calls one of the low level functions exactTestDoubleTail, exactTestBetaApprox, exactTestBySmallP or exactTestByDeviance to do the p-value computation. The low level functions all assume that the libraries have been normalized to have the same size, i.e., to have the same expected column sum under the null hypothesis. exactTest equalizes the library sizes using equalizeLibSizes before calling the low level functions.

The functions exactTestDoubleTail, exactTestBySmallP and exactTestByDeviance correspond to different ways to define the two-sided rejection region when the two groups have different numbers of samples. exactTestBySmallP implements the method of small probabilities as proposed by Robinson and Smyth (2008). This method corresponds exactly to binomTest as the dispersion approaches zero, but gives poor results when the dispersion is very large. exactTestDoubleTail computes two-sided p-values by doubling the smaller tail probability. exactTestByDeviance uses the deviance goodness of fit statistics to define the rejection region, and is therefore equivalent to a conditional likelihood ratio test.

Note that rejection.region="smallp" is no longer recommended. It is preserved as an option only for backward compatiblity with early versions of edgeR. rejection.region="deviance" has good theoretical statistical properties but is relatively slow to compute. rejection.region="doubletail" is just slightly more conservative than rejection.region="deviance", but is recommended because of its much greater speed. For general remarks on different types of rejection regions for exact tests see Gibbons and Pratt (1975).

exactTestBetaApprox implements an asymptotic beta distribution approximation to the conditional count distribution. It is called by the other functions for rows with both group counts greater than big.count.

Value

exactTest produces an object of class DGEExact containing the following components:

The low-level functions, exactTestDoubleTail etc, produce a numeric vector of genewise p-values, one for each row of y1 and y2.

Author(s)

Mark Robinson, Davis McCarthy, Gordon Smyth

References

Robinson MD and Smyth GK (2008). Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics, 9, 321-332.

Gibbons, JD and Pratt, JW (1975). P-values: interpretation and methodology. The American Statistician 29, 20-25.

See Also

equalizeLibSizes, binomTest

Examples

# generate raw counts from NB, create list object
y <- matrix(rnbinom(80,size=1/0.2,mu=10),nrow=20,ncol=4)
d <- DGEList(counts=y, group=c(1,1,2,2), lib.size=rep(1000,4))

de <- exactTest(d, dispersion=0.2)
topTags(de)

# same p-values using low-level function directly
p.value <- exactTestDoubleTail(y[,1:2], y[,3:4], dispersion=0.2)
sort(p.value)[1:10]

Description

Expand scalar or vector to a matrix.

Usage

expandAsMatrix(x, dim=NULL, byrow=TRUE)

Arguments

Details

This function expands a scalar, row/column vector or CompressedMatrix to be a matrix of dimensions dim. It is used internally in edgeR to convert offsets, weights and other values to a matrix for consistent handling. If dim is NULL, the function is equivalent to calling as.matrix(x).

If x is a vector, its length must match one of the output dimensions. The matrix will then be filled by repeating the matrix across the matching dimension. For example, if length(x)==dim[1], the matrix will be filled such that each row contains x. If both dimensions match, filling is determined by byrow, with filling by rows as the default.

If x CompressedMatrix object, the size of any non-repeated dimensions must be consistent with corresponding output dimension. The byrow argument will be ignored as the repeat specifications will dictate how expansion should be performed. See ?CompressedMatrix for more details.

Value

Numeric matrix of dimension dim.

Author(s)

Gordon Smyth

Examples

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

Description

Converts the list output by Rsubread::featureCounts to a DGEList object.

Usage

featureCounts2DGEList(x)

Arguments

Details

Rsubread's featureCounts function counts reads by features (typically exons or genomic intervals) or meta-features (typically genes). It may also count reads crossing exon-exon junctions or reads internal to exons. This function assembles all the read counts output by featureCounts into an DGEList, ensuring unique row.names where appropriate. The proportion of reads assigned to features is also stored.

Value

A DGEList data object.

Author(s)

Gordon Smyth.

See Also

DGEList-class

Description

Determine which genes have sufficiently large counts to be retained in a statistical analysis.

Usage

## S3 method for class 'DGEList'
filterByExpr(y, design = NULL, group = NULL, lib.size = NULL, ...)
## S3 method for class 'SummarizedExperiment'
filterByExpr(y, design = NULL, group = NULL, lib.size = NULL, ...)
## Default S3 method:
filterByExpr(y, design = NULL, group = NULL, lib.size = NULL,
             min.count = 10, min.total.count = 15, large.n = 10, min.prop = 0.7, ...)

Arguments

Details

This function implements the filtering strategy that was described informally by Chen et al (2016). Roughly speaking, the strategy keeps genes that have at least min.count reads in a worthwhile number samples.

More precisely, the filtering keeps genes that have CPM >= CPM.cutoff in MinSampleSize samples, where CPM.cutoff = min.count/median(lib.size)*1e6 and MinSampleSize is the smallest group sample size or, more generally, the minimum inverse leverage computed from the design matrix.

If all the group samples sizes are large, then the above filtering rule is relaxed slightly. If MinSampleSize > large.n, then genes are kept if CPM >= CPM.cutoff in k samples where k = large.n + (MinSampleSize - large.n) * min.prop. This rule requires that genes are expressed in at least min.prop * MinSampleSize samples, even when MinSampleSize is large.

In addition, each kept gene is required to have at least min.total.count reads across all the samples.

Value

Logical vector of length nrow(y) indicating which rows of y to keep in the analysis.

Author(s)

Gordon Smyth

References

Chen Y, Lun ATL, and Smyth, GK (2016). From reads to genes to pathways: differential expression analysis of RNA-Seq experiments using Rsubread and the edgeR quasi-likelihood pipeline. F1000Research 5, 1438. https://f1000research.com/articles/5-1438

Examples

## Not run: 
keep <- filterByExpr(y, design)
y <- y[keep,]

## End(Not run)

Description

getCounts(y) returns the matrix of read counts y$counts.

getOffset(y) returns offsets for the log-linear predictor account for sequencing depth and possibly other normalization factors. Specifically it returns the matrix y$offset if it is non-null, otherwise it returns the log product of lib.size and norm.factors from y$samples.

getDispersion(y) returns the most complex dispersion estimates (common, trended or genewise) found in y.

Usage

getCounts(y) 
getOffset(y) 
getDispersion(y)

Arguments

Value

getCounts returns the matrix of counts. getOffset returns a numeric matrix or vector. getDispersion returns vector of dispersion values.

Author(s)

Mark Robinson, Davis McCarthy, Gordon Smyth

See Also

DGEList-class

Examples

# generate raw counts from NB, create list object
y <- matrix(rnbinom(20,size=5,mu=10),5,4)
d <- DGEList(counts=y, group=c(1,1,2,2), lib.size=1001:1004)
getCounts(d)
getOffset(d)
d <- estimateCommonDisp(d)
getDispersion(d)

Description

Extract effective (normalized) library sizes.

Usage

## Default S3 method:
getNormLibSizes(y, log = FALSE, ...)

Arguments

Details

This function extracts normalized library sizes, equal to the original library sizes multiplied by the corresponding normalization factors, from an edgeR data object or fitted model object.

If the object contains a row-specific offsets (i.e., a non-sparse matrix of offsets), then the offsets for the first row are returned.

Value

A numeric matrix of effective (normalized) library sizes. If log=TRUE, then natural log values are returned, equal to library size offsets for a NB log-linear model.

Author(s)

Gordon Smyth

See Also

normLibSizes

Examples

ngenes <- 100
nsamples <- 4
y <- DGEList(counts=matrix(rnbinom(ngenes*nsamples,size=1,mu=10),ngenes,nsamples))
y <- normLibSizes(y)
data.frame(y$samples, eff.lib.size=getNormLibSizes(y))

Description

Returns the lib.size component of the samples component of DGEList object multiplied by the norm.factors component

Usage

getPriorN(y, design=NULL, prior.df=20)

Arguments

Details

When estimating genewise dispersion values using estimateTagwiseDisp or estimateGLMTagwiseDisp we need to decide how much weight to give to the common parameter likelihood in order to smooth (or stabilize) the dispersion estimates. The best choice of value for the prior.n parameter varies between datasets depending on the number of samples in the dataset and the complexity of the model to be fit. The value of prior.n should be inversely proportional to the residual degrees of freedom. We have found that choosing a value for prior.n that is equivalent to giving the common parameter likelihood 20 degrees of freedom generally gives a good amount of smoothing for the genewise dispersion estimates. This function simply recommends an appropriate value for prior.n—to be used as an argument for estimateTagwiseDisp or estimateGLMTagwiseDisp—given the experimental design at hand and the chosen prior degrees of freedom.

Value

getPriorN returns a numeric scalar

Author(s)

Davis McCarthy, Gordon Smyth

See Also

DGEList for more information about the DGEList class. as.matrix.DGEList.

Examples

# generate raw counts from NB, create list object
y<-matrix(rnbinom(20,size=1,mu=10),nrow=5)
d<-DGEList(counts=y,group=rep(1:2,each=2),lib.size=rep(c(1000:1001),2))
getPriorN(d)

Description

Gini index for each column of a matrix.

Usage

gini(x)

Arguments

Details

The Gini coefficient or index is a measure of inequality or diversity. It is zero if all the values of x are equal. It reaches a maximum value of 1/nrow(x) when all values are zero except for one.

The Gini index is only interpretable for non-negative quantities. It is not meaningful if x contains negative values.

Value

Numeric vector of length ncol(x).

Author(s)

Gordon Smyth

References

https://en.wikipedia.org/wiki/Gini_coefficient.

Examples

x <- matrix(rpois(20,lambda=5),10,2)
gini(x)

Description

Fit a negative binomial generalized log-linear model to the read counts for each gene.

Usage

## Default S3 method:
glmFit(y, design=NULL, dispersion=NULL, offset=NULL, lib.size=NULL, weights=NULL,
       prior.count=0.125, start=NULL, ...)
## S3 method for class 'DGEList'
glmFit(y, design=NULL, dispersion=NULL, prior.count=0.125, start=NULL, ...)
## S3 method for class 'SummarizedExperiment'
glmFit(y, design=NULL, dispersion=NULL, prior.count=0.125, start=NULL, ...)

Arguments

Details

Implements generalized linear model (GLM) methods developed by McCarthy et al (2012). Specifically, glmFit fits genewise negative binomial GLMs, all with the same design matrix but possibly different dispersions, offsets and weights. When the design matrix defines a one-way layout, or can be re-parametrized to a one-way layout, the GLMs are fitting very quickly using mglmOneGroup. Otherwise the default fitting method, implemented in mglmLevenberg, uses a Fisher scoring algorithm with Levenberg-style damping.

Positive prior.count values cause the returned coefficients to be shrunk in such a way that fold-changes between the treatment conditions are decreased and infinite fold-changes are avoided (Phipson, 2013). Larger prior.count values cause more shrinkage. Coefficient shrinkage does not affect the likelihood ratio tests or p-values.

Value

An object of class DGEGLM containing components counts, samples, genes and abundance from y plus the following new components:

Author(s)

Davis McCarthy, Gordon Smyth, Yunshun Chen, Aaron Lun

References

McCarthy DJ, Chen Y, Smyth GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297. doi:10.1093/nar/gks042

Phipson B (2013). Empirical bayes modelling of expression profiles and their associations. Ph.D. thesis, Department of Mathematics and Statistics, The University of Melbourne. http://hdl.handle.net/11343/38162.

Chen Y, Chen L, Lun ATL, Baldoni PL, Smyth GK (2024). edgeR 4.0: powerful differential analysis of sequencing data with expanded functionality and improved support for small counts and larger datasets. bioRxiv 2024.01.21.576131. doi:10.1101/2024.01.21.576131

See Also

Low-level computations are done by mglmOneGroup or mglmLevenberg.

topTags displays results from glmLRT.

Examples

nlibs <- 3
ngenes <- 100
dispersion.true <- 0.1

# Make first gene respond to covariate x
x <- 0:2
design <- model.matrix(~x)
beta.true <- cbind(Beta1=2,Beta2=c(2,rep(0,ngenes-1)))
mu.true <- 2^(beta.true %*% t(design))

# Generate count data
y <- rnbinom(ngenes*nlibs,mu=mu.true,size=1/dispersion.true)
y <- matrix(y,ngenes,nlibs)
colnames(y) <- c("x0","x1","x2")
rownames(y) <- paste("gene",1:ngenes,sep=".")
d <- DGEList(y)

# Normalize
d <- normLibSizes(d)

# Fit the NB GLMs
fit <- glmFit(d, design, dispersion=dispersion.true)

# Likelihood ratio tests for trend
results <- glmLRT(fit, coef=2)
topTags(results)

Description

Given genewise generalized linear model fits, conduct likelihood ratio tests for a given coefficient or coefficient contrast.

Usage

glmLRT(glmfit, coef=ncol(glmfit$design), contrast=NULL)

Arguments

Details

Using genewise GLMs from glmFit, glmLRT conducts likelihood ratio tests for one or more coefficients in the linear model. If contrast is NULL, the null hypothesis is that all the coefficients specified by coef are equal to zero. If contrast is non-NULL, then the null hypothesis is that the specified contrasts of the coefficients are equal to zero. For example, contrast = c(0,1,-1), assuming there are three coefficients, would test the hypothesis that the second and third coefficients are equal. If contrast is a matrix, then each column is a contrast and the null hypothesis is that all the contrasts are equal to zero.

Value

An object of class DGELRT with the same components as for glmFit plus the following:

The data frame table contains the following columns:

Author(s)

Gordon Smyth, Davis McCarthy, Yunshun Chen

References

McCarthy DJ, Chen Y, Smyth GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297. doi:10.1093/nar/gks042

See Also

glmFit fits the genewise GLMs that are input to glmLRT.

topTags displays results from glmLRT.

Examples

# See glmFit for an example that includes glmLRT

Description

Fit a negative binomial generalized log-linear model to the read counts for each gene Estimate the genewise quasi-dispersions with empirical Bayes moderation.

Usage

## Default S3 method:
glmQLFit(y, design = NULL, dispersion = NULL, offset = NULL, lib.size = NULL,
        weights = NULL, abundance.trend = TRUE, AveLogCPM = NULL,
        covariate.trend = NULL, robust = FALSE, winsor.tail.p = c(0.05, 0.1),
        legacy = FALSE, top.proportion = NULL, keep.unit.mat=FALSE, ...)
## S3 method for class 'DGEList'
glmQLFit(y, design = NULL, dispersion = NULL, abundance.trend = TRUE,
        robust = FALSE, winsor.tail.p = c(0.05, 0.1),
        legacy = FALSE, top.proportion = NULL, keep.unit.mat=FALSE, ...)
## S3 method for class 'SummarizedExperiment'
glmQLFit(y, design = NULL, dispersion = NULL, abundance.trend = TRUE,
        robust = FALSE, winsor.tail.p = c(0.05, 0.1),
        legacy = FALSE, top.proportion = NULL, keep.unit.mat=FALSE, ...)