DMLtest {DSS} | R Documentation

## Function to perform statistical test of differntially methylated loci (DML) for two group comparisons of bisulfite sequencing (BS-seq) data.

### Description

This function takes a BSseq object and two group labels, then perform statistical tests for differntial methylation at each CpG site.

### Usage

    DMLtest(BSobj, group1, group2, equal.disp = FALSE, smoothing = FALSE,
            smoothing.span = 500, BPPARAM = bpparam())

### Arguments

`BSobj` | An object of BSseq class for the BS-seq data.
`group1, group2` | Vectors of sample names or indexes for the two groups to be tested. See more description in details.
`equal.disp` | A flag to indicate whether the dispersion in two groups are deemed equal. Default is FALSE, and the dispersion shrinkages are performed on two conditions independently.
`smoothing` | A flag to indicate whether to apply smoothing in estimating mean methylation levels.

`smoothing.span` | The size of smoothing window, in basepairs. Default is 500.
`BPPARAM` | An optional BiocParallelParam instance determining the parallel back-end to be used. See sections 'Parallelization' for details.

### Details

This is the core function for DML/DMR detection. Tests are performed at each CpG site under the null hypothesis that two groups means are equal. There is an option for applying smoothing or not in estimating mean methylation levels. We recommend to use smoothing=TRUE for whole-genome BS-seq data, and smoothing=FALSE for sparser data such like from RRBS or hydroxyl-methylation data (TAB-seq). If there is not biological replicate, smoothing=TRUE is required. See "Single replicate" section for details.

The BS-seq count data are modeled as Beta-Binomial distribution, where the biological variations are captured by the dispersion parameter. The dispersion parameters are estimated through a shrinakge estimator based on a Bayesian hierarchical model. Then a Wald test is performed at each CpG site.

Due to the differences in coverages, some CpG sites are not covered in both groups, and the test cannot be performed. Those loci will be ignored in test and results will be "NA".

### Value

A data frame with each row corresponding to a CpG site. Rows are sorted by chromosome number and genomic coordinates. The columns include:

`chr` | Chromosome number.
`pos` | Genomic coordinates.
`mu1, mu2` | Mean methylations of two groups.
`diff` | Difference of mean methylations of two groups.
`diff.se` | Standard error of the methylation difference.
`stat` | Wald statistics.
`pval` | P-values. This is obtained from normal distribution.
`fdr` | False discovery rate.

### Single replicate

When there is no biological replicate in one or both treatment groups, users can either (1) specify equal.disp=TRUE, which assumes both groups have the same dispersion, then the data from two groups are combined and used as replicates to estimate dispersion; or (2) specify smoothing=TRUE, which uses the smoothed means (methylation levels) to estimate dispersions via a shrinkage estimator. This smoothing procedure uses data from neighboring CpG sites as "pseudo-replicate" for estimating biological variance.

### Parallelization

The shrinkage estimation for dispersion is the most computational component in DML testing. We now uses the 'BiocParallel' package to implement parallelization, which provides significant computational improvements.

All parallelization options are controlled via the 'BPPARAM' argument. BiocParallel provides params for SerialParam (Unix, Mac, Windows), MulticoreParam (Unix and Mac), SnowParam (Unix, Mac, and Windows, limited to single-machine clusters), and BatchJobsParam (Unix, Mac, Windows). By default, 'BPPARAM' takes value from 'bpparam()'. Example section provides some codes to generate user-specified BPPARAM. For more details, please read the 'BiocParallel' documentation.

### Estimating mean methylation levels

When smoothing=FALSE, the mean methylation levels are estimated based on the ratios of methylated and total read counts, and the spatial correlations among nearby CpG sites are ignored. When smoothing=TRUE, smoothing based on moving average or the BSmooth method is used to estimate the mean methylaion level at each site. Moving average is recommended because it is much faster than BSmooth, and the results are reasonable similar in terms of mean estimation, dispersion estimation, and DMR calling results.

### Author(s)

Hao Wu <hao.wu@emory.edu>

### See Also

makeBSseqData, callDML, callDMR

### Examples

    ## Not run:
    require(bsseq)

    ## first read in methylation data.
    path <- file.path(system.file(package="DSS"), "extdata")
    dat1.1 <- read.table(file.path(path, "cond1_1.txt"), header=TRUE)
    dat1.2 <- read.table(file.path(path, "cond1_2.txt"), header=TRUE)
    dat2.1 <- read.table(file.path(path, "cond2_1.txt"), header=TRUE)
    dat2.2 <- read.table(file.path(path, "cond2_2.txt"), header=TRUE)

    ## make BSseq objects
    BSobj <- makeBSseqData( list(dat1.1, dat1.2, dat2.1, dat2.2),
      c("C1","C2", "N1", "N2") )

    ##  DML test without smoothing
    dmlTest <- DMLtest(BSobj, group1=c("C1", "C2"), group2=c("N1", "N2"))
    head(dmlTest)

    ## For whole-genome BS-seq data, perform DML test with smoothing
    require(bsseqData)
    data(BS.cancer.ex)
    ## take a small portion of data and test
    BSobj <- BS.cancer.ex[10000:15000,]
    dmlTest <- DMLtest(BSobj, group1=c("C1", "C2", "C3"), group2=c("N1","N2","N3"),
                       smoothing=TRUE, smoothing.span=500)
    head(dmlTest)

    ## Examples for Parallelization
    ## use single core - this has not parallelization
    single = MulticoreParam(workers=1, progressbar=TRUE)
    dmlTest <- DMLtest(BSobj, group1=c("C1", "C2"), group2=c("N1", "N2"),
                       BPPARAM=single)

    ## use 4 cores
    mParam = MulticoreParam(workers=4, progressbar=TRUE)
    dmlTest <- DMLtest(BSobj, group1=c("C1", "C2"), group2=c("N1", "N2"),
                       BPPARAM=mParam)

    ## use SnowParam, this will work on Windows
    snow <- SnowParam(workers = numCores)
    dmlTest <- DMLtest(BSobj, group1=c("C1", "C2"), group2=c("N1", "N2"),
                       BPPARAM=snow)

    ## End(Not run)

[Package DSS version 2.40.0 Index]