eBayes {limma} | R Documentation

## Empirical Bayes Statistics for Differential Expression

### Description

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

### Usage

    eBayes(fit, proportion = 0.01, stdev.coef.lim = c(0.1,4),
           trend = FALSE, robust = FALSE, winsor.tail.p = c(0.05,0.1))
    treat(fit, lfc = log2(1.2), trend = FALSE, robust = FALSE, winsor.tail.p = c(0.05,0.1))

### Arguments

`fit` | an `MArrayLM` fitted model object produced by `lmFit` or `contrasts.fit`. For `ebayes` only, `fit` can alternatively be an unclassed list produced by `lm.series`, `gls.series` or `mrlm` containing components `coefficients`, `stdev.unscaled`, `sigma` and `df.residual`.
`proportion` | numeric value between 0 and 1, assumed proportion of genes which are differentially expressed
`stdev.coef.lim` | numeric vector of length 2, assumed lower and upper limits for the standard deviation of log2-fold-changes for differentially expressed genes
`trend` | logical, should an intensity-trend be allowed for the prior variance? Default is that the prior variance is constant.
`robust` | logical, should the estimation of `df.prior` and `var.prior` be robustified against outlier sample variances?
`winsor.tail.p` | numeric vector of length 1 or 2, giving left and right tail proportions of `x` to Winsorize. Used only when `robust=TRUE`.
`lfc` | the minimum log2-fold-change that is considered scientifically meaningful

### Details

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

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

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

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

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

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

Note that the `lfc` testing threshold used by `treat` to the define the null hypothesis is not the same as a log2-fold-change cutoff, as the observed log2-fold-change needs to substantially larger than `lfc` for the gene to be called as significant. In practice, modest values for `lfc` such as `log2(1.1)`, `log2(1.2)` or `log2(1.5)` are usually the most useful. In practice, setting `lfc=log2(1.2)` or `lfc=log2(1.5)` will usually cause most differentially expressed genes to have estimated fold-changes of 2-fold or greater, depending on the sample size and precision of the experiment.

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

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

### Value

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

`t` | numeric vector or matrix of moderated t-statistics
`p.value` | numeric vector of p-values corresponding to the t-statistics
`s2.prior` | estimated prior value for `sigma^2`. A vector if `covariate` is non-`NULL`, otherwise a scalar.
`df.prior` | degrees of freedom associated with `s2.prior`
`df.total` | numeric vector of total degrees of freedom associated with t-statistics and p-values. Equal to `df.prior+df.residual` or `sum(df.residual)`, whichever is smaller.
`s2.post` | numeric vector giving the posterior values for `sigma^2`
`lods` | numeric vector or matrix giving the log-odds of differential expression (natural log scale).
`var.prior` | estimated prior value for the variance of the log2-fold-change for differentially expressed gene
`F` | numeric vector of moderated F-statistics for testing all contrasts defined by the columns of `fit` simultaneously equal to zero
`F.p.value` | numeric vector giving p-values corresponding to `F`

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

### Note

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

### Author(s)

Gordon Smyth and Davis McCarthy

### References

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

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

McCarthy, D. J., and Smyth, G. K. (2009). Testing significance relative to a fold-change threshold is a TREAT. Bioinformatics 25, 765-771. http://bioinformatics.oxfordjournals.org/content/25/6/765

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

Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, Article 3. http://www.statsci.org/smyth/pubs/ebayes.pdf

### See Also

`squeezeVar`, `fitFDist`, `tmixture.matrix`, `plotSA`.

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

### Examples

    #  See also lmFit examples

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

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

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

    # Treat relative to a 10% fold-change
    tfit <- treat(fit,lfc=log2(1.1))
    topTreat(tfit,coef=2)

[Package limma version 3.44.3 Index]