Overview of the MultiAssayExperiment class

empty <- MultiAssayExperiment()

Components of the MultiAssayExperiment

Elist: experiment data

The Elist slot and class is the container workhorse for the MultiAssayExperiment class. It contains all the experiment data. It inherits from class S4Vectors::SimpleList with one element/component per experiment type.


The elements of the Elist can contain ID-based and range-based data. Requirements for all classes in the Elist are listed in the API, see API() for details. These familiar base and Bioconductor classes are supported:

Class requirements within Elist container

The datasets contained in elements of the Elist must have:

The column names correspond to samples, and are used to match assay data to specimen metadata stored in pData.

The row names can correspond to a variety of features in the data including but not limited to gene names, probe IDs, proteins, and named ranges.


Classes contained in the Elist must support the following list of methods:

pData: primary data

The MultiAssayExperiment keeps one set of "primary" metadata that describes the 'biological unit' which can refer to specimens, experiment subject, patients, etc. In this tutorial, we will refer to each experiment subject as a patient.

pData slot requirements

The pData dataset should be of class DataFrame but can accept a data.frame class object that will subsequently be coerced.

In order to relate metadata of the biological unit, the row names of the pData dataset must contain patient identifiers.

patient.data <- data.frame(sex=c("M", "F", "M", "F"),
    row.names=c("Jack", "Jill", "Bob", "Barbara"))

sampleMap: relating pData to multiple assays

sampleMap is a DataFrame that provides a map between the "primary" data (pData) and the experimental assays:


The sampleMap provides an unambiguous map from every experimental observation to one and only one row in pData. It is, however, permissible for a row of pData to be associated with multiple experimental observations or no observations at all. In other words, there is a "many-to-one" mapping from experimental observations to rows of pData, and a "one-to-any-number" mapping from rows of pData to experimental observations.

sampleMap structure

pData has three columns, with the following column names:

  1. primary provides the "primary" sample names. All values in this column must also be present in the rownames of pData(MultiAssayExperiment). In this example, allowable values in this column are "Jack", "Jill", "Barbara", and "Bob".

  2. assay provides the sample names used by experimental datasets, which in practice are often different than the primary sample names. For each assay, all column names must be found in this column. Otherwise, those assays would be orphaned: it would be impossible to match them up to samples in the overall experiment. As mentioned above, duplicated values are allowed, to represent replicates with the same overall experiment-level annotation.

  3. assayname provides the names of the different experiments / assays performed. These are user-defined, with the only requirement that the names of the Elist, where the experimental assays are stored, must be contained in this column.

Instances where sampleMap isn't provided

If each assay uses the same colnames (i.e., if the same sample identifiers are used for each experiment), a simple list of these datasets is sufficient for the MultiAssayExperiment() constructor function. It is not necessary for them to have the same rownames or colnames:

exprss1 <- matrix(rnorm(16), ncol = 4,
        dimnames = list(sprintf("ENST00000%i", sample(288754:290000, 4)),
                c("Jack", "Jill", "Bob", "Bobby")))
exprss2 <- matrix(rnorm(12), ncol = 3,
        dimnames = list(sprintf("ENST00000%i", sample(288754:290000, 4)),
                c("Jack", "Jane", "Bob")))
doubleExp <- list("methyl 2k"  = exprss1, "methyl 3k" = exprss2)
simpleMultiAssay <- MultiAssayExperiment(Elist=doubleExp)

In the above example, the user did not provide the pData argument so the constructor function filled it with a trivial DataFrame:


But the pData can be provided. Here, note that any assay sample (column) that cannot be mapped to a corresponding row in the provided pData gets dropped. This is part of ensuring internal validity of the MultiAssayExperiment.

simpleMultiAssay2 <- MultiAssayExperiment(Elist=doubleExp, pData=patient.data)


Can be of ANY class, for storing study-wide metadata, such as citation information. For an empty MultiAssayExperiment object, it is NULL.

class(metadata(empty)) # NULL (class "ANY")

Creating a MultiAssayExperiment object: a rich example

In this section we demonstrate all core supported data classes, using different sample ID conventions for each assay, with primary pData. The some supported classes such as, matrix, ExpressionSet, SummarizedExperiment, RangedSummarizedExperiment, and RangedRaggedAssay.

Create toy datasets demonstrating all supported data types

We have three matrix-like datasets. First let's say expression data, which in this example we represent as an ExpressionSet:

(arraydat <- matrix(seq(101, 108), ncol=4,
                    dimnames=list(c("ENST00000294241", "ENST00000355076"),
                                  c("array1", "array2", "array3", "array4"))))
arraypdat <- as(data.frame(slope53=rnorm(4),
                           row.names=c("array1", "array2", "array3",
                                       "array4")), "AnnotatedDataFrame")
exprdat <- ExpressionSet(assayData=arraydat, phenoData=arraypdat)

The following map matches pData sample names to exprdata sample names. Note that row orders aren't initially matched up, and this is OK.

(exprmap <- data.frame(primary=rownames(patient.data)[c(1, 2, 4, 3)],
                       assay=c("array1", "array2", "array3", "array4"),
                       stringsAsFactors = FALSE))

Now methylation data, which we will represent as a matrix. It uses gene identifiers also, but measures a partially overlapping set of genes. For fun, let's store this as a simple matrix. Also, it contains a replicate for one of the patients.

(methyldat <-
   matrix(1:10, ncol=5,
          dimnames=list(c("ENST00000355076", "ENST00000383706"),
                        c("methyl1", "methyl2", "methyl3",
                          "methyl4", "methyl5"))))

The following map matches pData sample names to methyldat sample names.

(methylmap <- data.frame(primary = c("Jack", "Jack", "Jill", "Barbara", "Bob"),
              assay = c("methyl1", "methyl2", "methyl3", "methyl4", "methyl5"),
              stringsAsFactors = FALSE))

Now we have a microRNA platform, which has no common identifiers with the other datasets, and which we also represent as a matrix. It is also missing data for Jill. Just for fun, let's use the same sample naming convention as we did for arrays.

(microdat <- matrix(201:212, ncol=3,
                    dimnames=list(c("hsa-miR-21", "hsa-miR-191",
                                    "hsa-miR-148a", "hsa-miR148b"),
                                  c("micro1", "micro2", "micro3"))))

And the following map matches pData sample names to microdat sample names.

(micromap <- data.frame(primary = c("Jack", "Barbara", "Bob"),
                        assay = c("micro1", "micro2", "micro3"),
                        stringsAsFactors = FALSE))

Let's include a RangedRaggedAssay, which is defined in this package and extends GRangesList. This is intended for data such as segmented copy number, which provide genomic ranges that may be different for each sample. We start with a GRangesList, which will later be converted automatically by the MultiAssayExperiment constructor function.

## completely encompasses ENST00000355076
gr1 <-
  GRanges(seqnames = "chr3", ranges = IRanges(58000000, 59502360),
          strand = "+", score = 5L, GC = 0.45)
## first is within ENST0000035076
gr2 <-
  GRanges(seqnames = c("chr3", "chr3"),
          ranges = IRanges(c(58493000, 3), width=9000),
          strand = c("+", "-"), score = 3:4, GC = c(0.3, 0.5))
gr3 <-
  GRanges(seqnames = c("chr1", "chr2"),
          ranges = IRanges(c(1, 4), c(3, 9)),
          strand = c("-", "-"), score = c(6L, 2L), GC = c(0.4, 0.1))
grl <- GRangesList("gr1" = gr1, "gr2" = gr2, "gr3" = gr3)
names(grl) <- c("snparray1", "snparray2", "snparray3")

The following data.frame matches pData sample to the GRangesList:

(rangemap <- data.frame(primary = c("Jack", "Jill", "Jill"),
                        assay = c("snparray1", "snparray2", "snparray3"),
                        stringsAsFactors = FALSE))

Finally, we create a dataset of class RangedSummarizedExperiment:

nrows <- 5; ncols <- 4
counts <- matrix(runif(nrows * ncols, 1, 1e4), nrows)
rowRanges <- GRanges(rep(c("chr1", "chr2"), c(2, nrows - 2)),
                     IRanges(floor(runif(nrows, 1e5, 1e6)), width=100),
                     strand=sample(c("+", "-"), nrows, TRUE),
                     feature_id=sprintf("ID\\%03d", 1:nrows))
names(rowRanges) <- letters[1:5]
colData <- DataFrame(Treatment=rep(c("ChIP", "Input"), 2),
                     row.names= c("mysnparray1", "mysnparray2",
                                  "mysnparray3", "mysnparray4"))
rse <- SummarizedExperiment(assays=SimpleList(counts=counts),
                            rowRanges=rowRanges, colData=colData)
(rangemap2 <-
   data.frame(primary = c("Jack", "Jill", "Bob", "Barbara"),
              assay = c("mysnparray1", "mysnparray2", "mysnparray3",
                        "mysnparray4"), stringsAsFactors = FALSE))

sampleMap creation

The MultiAssayExperiment constructor function can create the sampleMap automatically if a single naming convention is used, but in this example it cannot because we used platform-specific sample identifiers (e.g. mysnparray1, etc). So we must provide an ID map that matches the samples of each experiment back to the pData, as a three-column data.frame or DataFrame with three columns named "primary", "assay", and "assayname". Here we start with a list:

listmap <- list(exprmap, methylmap, micromap, rangemap, rangemap2)
names(listmap) <- c("Affy", "Methyl 450k", "Mirna", "CNV gistic", "CNV gistic2")

and use the convenience function listToMap to convert the list of data.frame objects to a valid object for the sampleMap:

dfmap <- listToMap(listmap)

Note, dfmap can be reverted to a list with another provided function:

mapToList(dfmap, "assayname")

Experimental data as a list()

Create an named list of experiments for the MultiAssay function. All of these names must be found within in the third column of dfmap:

objlist <- list("Affy" = exprdat, "Methyl 450k" = methyldat,
                "Mirna" = microdat, "CNV gistic" = grl, "CNV gistic2" = rse)

Creation of the MultiAssayExperiment class object

We recommend using the MultiAssayExperiment() constructor function:

myMultiAssay <- MultiAssayExperiment(objlist, patient.data, dfmap)

The following extractor functions can be used to get extract data from the object:


Note that the Elist class extends the SimpleList class to add some validity checks specific to MultiAssayExperiment. It can be used like a list.

Helper function to create a MultiAssayExperiment object

The PrepMultiAssay function helps diagnose common problems when creating a MultiAssayExperiment object. It provides error messages and/or warnings in instances where names (either colnames or Elist element names) are inconsistent with those found in the sampleMap. Input arguments are the same as those in the MultiAssayExperiment (i.e., Elist, pData, sampleMap). The resulting output of the PrepMultiAssay function is a list of inputs including a "drops" element for names that were not able to be matched.

Instances where Elist is created without names will prompt an error from PrepMultiAssay. Named Elist elements are essential for checks in MultiAssayExperiment.

objlist3 <- objlist
(names(objlist3) <- NULL)
try(PrepMultiAssay(objlist3, patient.data, dfmap)$Elist)

Non-matching names may also be present in the Elist elements and the "assayname" column of the sampleMap. If names only differ by case and are identical and unique, names will be standardized to lower case and replaced.

names(objlist3) <- toupper(names(objlist))
unique(dfmap[, "assayname"])
PrepMultiAssay(objlist3, patient.data, dfmap)$Elist

When colnames in the Elist cannot be matched back to the primary data (pData), these will be dropped and added to the drops element.

exampleMap <- sampleMap(simpleMultiAssay2)
sapply(doubleExp, colnames)
PrepMultiAssay(doubleExp, patient.data, exampleMap)$drops

A similar operation is performed for checking "primary" sampleMap names and pData rownames. In this example, we add a row corresponding to "Joe" that does not have a match in the experiment data.

exMap <- rbind(dfmap,
               DataFrame(primary = "Joe",
                         assay = "Joe",
                         assayname = "New methyl"))
PrepMultiAssay(objlist, patient.data, exMap)$drops

To create a MultiAssayExperiment from the results of the PrepMultiAssay function, take each corresponding element from the resulting list and enter them as arguments to the MultiAssayExperiment constructor function.

prepped <- PrepMultiAssay(objlist, patient.data, exMap)
preppedMulti <- MultiAssayExperiment(prepped$Elist, prepped$pData, prepped$sampleMap)

RangedRaggedAssay class

Note that the GRangesList got converted to a RangedRaggedAssay, a class intended for data such as segmented copy number that is provides different genomic ranges for each sample. RangedRaggedAssay is defined by this package and inherits from GRangesList:

getMethod("colnames", "RangedRaggedAssay")

It has some additional methods that are required for any data class contained in a MultiAssayExperiment:


One of the requirements for the assay method (specifically for this RangedRaggedAssay Elist element) is that the metadata have a score column from which to obtain values for the resulting assay matrix. Here we add ficticious values to such column contained within list elements. See assay,RangedRaggedAssay,ANY-method documentation.

metadata(Elist(myMultiAssay)[[4]]) <- list(snparray1 = DataFrame(score = 1),
                                           snparray2 = DataFrame(score = 1),
                                           snparray3 = DataFrame(score = 3))
assay(Elist(myMultiAssay)[[4]], background = 2)

Integrated subsetting across experiments

The core functionality of MultiAssayExperiment is to allow subsetting by assay, rownames, and colnames, across all experiments simultaneously while guaranteeing continued matching of samples.

Subsetting samples / columns

Experimental samples are stored in the rows of pData but the columns of elements of Elist, so when we refer to subsetting by columns, we are referring to columns of the experimental assays. Subsetting by samples / columns will be more obvious after recalling the pData:


Subsetting by samples identifies the selected samples in rows of the pData DataFrame, then selects all columns of the Elist corresponding to these rows. Here we use an integer to keep the first two rows of pData, and all experimental assays associated to those two primary samples:

subsetByColumn(myMultiAssay, 1:2)

Note that the above operation keeps different numbers of columns / samples from each assay, reflecting the reality that some samples may not have been assayed in all experiments, and may have replicates in some.

Subsetting the primary identifiers using a character vector corresponding to some rownames of pData returns the same result:

subsetByColumn(myMultiAssay, c("Jack", "Jill"))

Columns can be subset using a logical:

malesMultiAssay <- subsetByColumn(myMultiAssay, pData(myMultiAssay)$sex=="M")

Note that selecting male patients from all assays could have been accomplished equivalently using the square bracket:

myMultiAssay[, pData(myMultiAssay)$sex=="M", ]

Finally, for special use cases you can exert detail control of which samples to select using a list or CharacterList, which is just a convenient form of a list containing character vectors.

allsamples <- colnames(myMultiAssay)

Now let's get rid of the Methyl 450k arrays 3-5, a couple different but equivalent ways:

allsamples[["Methyl 450k"]] <- allsamples[["Methyl 450k"]][-3:-5]
myMultiAssay[, as.list(allsamples), ]

Subsetting assays

You can select certain assays / experiments using subset, by providing a character, logical, or integer vector. An example using character:

subsetByAssay(myMultiAssay, c("Affy", "CNV gistic"))

Examples using logical and integer:

is.cnv = grepl("CNV", names(Elist(myMultiAssay)))
subsetByAssay(myMultiAssay, is.cnv)
subsetByAssay(myMultiAssay, which(is.cnv))

subsetByRow, subsetByColumn, and subsetByAssay are endogenous operations, in that it always returns another MultiAssayExperiment object. Use assay(myMultiAssay) to retrieve the experimental data in an ordinary list of datasets as their original classes.

Subsetting rows (features) by IDs, integers, or logicals

Rows of the assays correspond to assay features or measurements, such as genes. Regardless of whether the assay is ID-based (e.g. matrix, ExpressionSet) or range-based (e.g. RangedSummarizedExperiment, RangedRaggedAssay), they can be subset using any of:

Again, this operation always returns a MultiAssayExperiment class, unless "drop=TRUE" is passed to subset, with any Elist element not containing the feature having zero rows.

For example, return a MultiAssayExperiment where Affy and Methyl 450k contain only ENST0000035076 row, and "Mirna" and "CNV gistic" have zero rows: (drop argument is set to TRUE by default)

featSubsetted0 <- subsetByRow(myMultiAssay, "ENST00000355076")

In the following, Affy ExpressionSet keeps both rows but with their order reversed, and Methyl 450k keeps only its second row.

featSubsetted <-
  subsetByRow(myMultiAssay, c("ENST00000355076", "ENST00000294241"))

Subsetting rows (features) by GenomicRanges

For MultiAssayExperiment objects containing range-based objects (currently RangedSummarizedExperiment and RangedRaggedAssay), these can be subset using a GRanges object, for example:

gr <- GRanges(seqnames = c("chr1"), strand = c("-", "+", "-"),
              ranges = IRanges(start = c(1, 4, 6), width = 3))

Now do the subsetting. The function doing the work here is IRanges::subsetByOverlaps - see its arguments for flexible types of subsetting by range. The first three arguments here are for subset, the rest passed on to IRanges::subsetByOverlaps through "...":

subsetted <- subsetByRow(myMultiAssay, gr, maxgap = 2L, type = "within")

Subsetting by square bracket [

The bracket method for the MultiAssayExperiment is equivalent but more compact than the subsetBy*() methods. The three positions within the bracket operator indicate rows, columns, and assays, respectively (pseudocode):

myMultiAssay[rows, columns, assays]

For example, to select the gene ENST00000355076:

myMultiAssay["ENST00000355076", , ]

The above operation works across all types of assays, whether ID-based (e.g. matrix, ExpressionSet, SummarizedExperiment) or range-based (e.g. RangedSummarizedExperiment, RangedRaggedAssay).

You can subset by rows, columns, and assays in a single bracket operation, and they will be performed in that order (rows, then columns, then assays):

myMultiAssay["ENST00000355076", 1:2, c("Affy", "Methyl 450k")]

Subsetting by character, integer, and logical

By columns - character, integer, and logical are all allowed, for example:

myMultiAssay[, "Jack", ]
myMultiAssay[, 1, ]
myMultiAssay[, c(TRUE, FALSE, FALSE, FALSE), ]

By assay - character, integer, and logical are allowed:

myMultiAssay[, , "Mirna"]
myMultiAssay[, , 3]
myMultiAssay[, , c(FALSE, FALSE, TRUE, FALSE, FALSE)]

the "drop" argument

Specify drop=FALSE to keep assays with zero rows or zero columns, e.g.:

myMultiAssay["ENST00000355076", , , drop=FALSE]

Using the default drop=TRUE, assays with no rows or no columns are removed:

myMultiAssay["ENST00000355076", , , drop=TRUE]

rownames and colnames

rownames and colnames return a CharacterList of rownames and colnames across all the assays. A CharacterList is just an alternative to list when each element contains a character vector, that provides a nice show method:


waldronlab/MultiAssayExperiment_Bioc2016 documentation built on May 27, 2017, 11:56 p.m.