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\begin{document}

\title{\bf Introduction to the Bioconductor marray package : Classes structure component}

\author{Sandrine Dudoit$^1$ and Yee Hwa Yang$^2$}

\maketitle

\begin{center}
1. Division of Biostatistics, University of California, Berkeley,
   \url{http://www.stat.berkeley.edu/~sandrine}
2. Department of Medicine, University of California, San Francisco,
   {\tt jean@biostat.berkeley.edu}\\
\end{center}

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\tableofcontents

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
\section{Overview}

This document provides a tutorial on the class structures used in the
{\tt marray} package.  The {\tt marray} packages contains basic class
definitions and associated methods for pre-- and post--normalization
intensity data for batches of arrays. To load the {\tt marray} package
in your R session, type {\tt library(marray)}.  As with any R package,
detailed information on functions, classes and methods can be obtained
in the help files. For instance, to view the help file for the class
{\tt marrayRaw} in a browser, use {\tt help.start()} followed by {\tt ?
marrayRaw} or alternately the dyadic {\tt class ? marrayRaw}.
Furthermore, se demonstrate the functionality of this collection of R
packages using gene expression data from the Swirl zebrafish
experiment. To load the Swirl dataset, use {\tt data(swirl)}, and to
view a description of the experiments and data, type {\tt ? swirl}.

Getting started:
<<eval=TRUE, echo=FALSE>>=
library("marray")
data(swirl)
@

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
\section{Object--oriented programming}\label{soo}

Microarray experiments generate large and complex multivariate datasets,
which contain textual information on probe sequences (e.g. gene names,
annotation, layout parameters) and mRNA target samples (e.g. description
of samples, protocols, hybridization and scanning conditions), in
addition to the primary fluorescence intensity data. Efficient and
coordinated access to these various types of data is an important aspect
of computing with microarray data. To facilitate the management of
microarray data at different stages of the analysis process, a
collection of microarray specific data structures or {\it classes} were
defined (see also the Bioconductor package {\tt Biobase} for microarray
classes and methods for normalized data). The packages rely on the
class/method mechanism provided by John Chambers' R {\tt methods}
package, which allows object--oriented programming in R. Broadly
speaking, {\it classes} reflect how we think of certain objects and what
information these objects should contain. Classes are defined in terms
of {\it slots} which contain the relevant data for the application at
hand. {\it Methods} define how a particular function should behave
depending on the class of its arguments and allow computations to be
adapted to particular classes, that is, data types. For example, a
microarray object should contain intensity data as well as information
on the probe sequences spotted on the array and the target samples
hybridized to it. Useful methods for microarray classes include
specializations of printing, subsetting, and plotting functions for the
types of data represented by these classes.  

The use of classes and methods greatly reduces the complexity of
handling microarray data, by automatically coordinating various sources
of information associated with microarray experiments. 

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
\section{Microarray classes}\label{sclasses}

The {\it raw data} from a microarray experiment are the image files
produced by the scanner; these are typically pairs of 16--bit tagged
image file format (TIFF) files, one for each fluorescent dye (images
usually range in size from a few megabytes (MB) to 10 or 20 MB for high
resolution scans). Image analysis is required to extract foreground and
background fluorescence intensity measurements for each spotted DNA
sequence. \\ 

Here, we begin our analysis of microarray data with the output files of
image processing packages such as {\tt GenePix} or {\tt Spot}. In what
follows, red and green background intensities are denoted by $R_b$ and
$G_b$, respectively, and red and green foreground intensities by $R_f$
and $G_f$, respectively. Background--corrected red and green
fluorescence intensities are denoted by $R$ and $G$, and $M$ denotes the
corresponding base--2 log--ratio, $M = \log_2 R/G$.  

\subsection{{\tt marrayLayout} class} 

The term {\it array layout} refers to the layout of DNA probe sequences
on the array, as determined by the printing process. In general, probe
sequences are spotted on a glass microscope slide using an arrayer which
has an $ngr \times ngc$ print--head, that is, a regular array of $ngr$
rows and $ngc$ columns of print--tips or pins. The resulting microarrays
are thus partitioned into an $ngr \times ngc$ {\it grid matrix}. The
terms {\it grid}, {\it sector}, and {\it print--tip--group} are used
interchangeably in the microarray literature. Each grid consists of an
$nsr \times nsc$ {\it spot matrix} that was printed with a single
print--tip. DNA probes are usually printed sequentially from a
collection of 384--well plates (or 96--well plates), thus, in some
sense, plates are proxies for time of printing. In addition, a number of
control probe sequences may be spotted on the array for normalization or
other calibration purposes. The term {\it array batch} is used to refer
to a collection of arrays with the same layout. Keeping track of spot
layout information is essential for quality assessment of fluorescent
intensity data and for normalization purposes.\\ 

Important layout parameters are the dimensions of the spot and grid
matrices, and, for each probe on the array, its grid matrix and spot
matrix coordinates. In addition, it is useful to keep track of gene
names, plate origin of the probes, and information on the spotted
control sequences (e.g. probe sequences which should have equal
abundance in the two target samples, such as housekeeping genes). The
class {\tt marrayLayout} was designed to keep track of these various
layout parameters and contains the following slots (the classes of the
slots are listed below the slot names) 

<<R>>=
getClassDef("marrayLayout")
@
\begin{description}
    \item{{\tt maNgr}:}{ Object of class "numeric", number of rows for the grid matrix.}
    \item{{\tt maNgc}:}{ Object of class "numeric", number of columns for the grid matrix.}
    \item{{\tt maNsr}:}{ Object of class "numeric",  number of rows for the spot matrices.} 
    \item{{\tt maNsc}:}{ Object of class "numeric", number of columns for the spot matrices.}
    \item{{\tt maNspots}:}{ Object of class "numeric", total number of
    spots on the array, equal to $maNgr \times maNgc \times maNsr \times
    maNsc$.} 
    \item{{\tt maSub}:}{ Object of class "logical", indicating which
    spots are currently being considered.} 
    \item{{\tt maPlate}:}{ Object of class "factor", recording the plate
    origin of the spotted probe sequences.}
    \item{{\tt maControls}:}{ Object of class "factor", recording the
    control status of the spotted probe sequences.} 
    \item{{\tt maNotes}:}{ Object of class "character",  any notes
    concerning the microarray layout, e.g., printing conditions.} 
\end{description}

In addition, a number of {\it methods} were defined to compute other
important layout parameters, such as print--tip, grid matrix, and spot
matrix coordinates: {\tt maPrintTip}, {\tt maGridRow}, {\tt maGridCol},
{\tt maSpotRow}, and {\tt maSpotCol} (see Section \ref{smethods}). No
slots were defined for these quantities for memory management reasons. 

\subsection{{\tt marrayInfo} class} 

Information on the target mRNA samples co--hybridized to the arrays is
stored in objects of class {\tt marrayInfo}. Such objects may include
the names of the arrays, the names of the Cy3 and Cy5 labeled samples,
notes on the hybridization and scanning conditions, and other textual
information. Descriptions of the spotted probe sequences (e.g. matrix of
gene names, annotation, notes on printing conditions) are also stored in
object of class {\tt marrayInfo}. The {\tt marrayInfo} class is not
specific to the microarray context and has the following definition 

<<R>>=
getClassDef("marrayInfo")
@

\subsection{{\tt marrayRaw} class}

 Pre--normalization intensity data for a batch of arrays are stored in
 objects of class {\tt marrayRaw}, which contain slots for the matrices
 of Cy3 and Cy5 background and foreground intensities ({\tt maGb}, {\tt
 maRb}, {\tt maGf}, {\tt maRf}), spot quality weights ({\tt maW}),
 layout parameters of the arrays ({\tt marrayLayout}), description of
 the probes spotted onto the arrays ({\tt maGnames}) and mRNA target
 samples hybridized to the arrays ({\tt maTargets}).  

<<R>>=
getClassDef("marrayRaw")
@

\begin{description}
  \item{{\tt maRf}:}{ Object of class "matrix", red foreground intensities, rows correspond to spotted probe sequences, columns to arrays in the batch.}
    \item{{\tt maGf}:}{ Object of class "matrix", green foreground intensities, rows correspond to spotted probe sequences, columns to arrays in the batch. }
    \item{{\tt maRb}:}{ Object of class "matrix", red background intensities, rows correspond to spotted probe sequences, columns to arrays in the batch. }
    \item{{\tt maGb}:}{ Object of class "matrix", green background intensities, rows correspond to spotted probe sequences, columns to arrays in the batch. }
    \item{{\tt maW}:}{ Object of class "matrix", spot quality weights, rows correspond to spotted probe sequences, columns to arrays in the batch.}
    \item{{\tt maLayout}:}{ Object of class "marrayLayout", layout parameters for cDNA microarrays.}
    \item{{\tt maGnames}:}{ Object of class "marrayInfo", description of spotted probe sequences.}
    \item{{\tt maTargets}:}{ Object of class "marrayInfo", description of target samples hybridized to the arrays.}
    \item{{\tt maNotes}:}{ Object of class "character", any notes concerning the microarray experiments, e.g. hybridization or scanning conditions.}
\end{description} 

\subsection{{\tt marrayNorm} class} 

Post--normalization intensity data are stored in similar objects of
class {\tt marrayNorm}. These objects store the normalized intensity
log--ratios {\tt maM}, the location and scale normalization values ({\tt
  maMloc} and {\tt maMscale}), and the average log--intensities ({\tt
  maA}). In addition, the {\tt marrayNorm} class has a slot for the
function call used to normalize the data, {\tt maNormCall}. For more
details on the creation of normalized microarray objects, the reader is
referred to the vignette for the {\tt marrayNorm} package. 

<<R>>=
getClassDef("marrayNorm")
@ 

\begin{description}
    \item{{\tt maA}:}{ Object of class "matrix", average log--intensities (base 2) $A$, rows correspond to spotted probe sequences, columns to arrays in the batch.}
    \item{{\tt maM}:}{ Object of class "matrix", intensity log--ratios (base 2) $M$, rows correspond to spotted probe sequences, columns to arrays in the batch.}
    \item{{\tt maMloc}:}{ Object of class "matrix", location normalization values, rows correspond to spotted probe sequences, columns to arrays in the batch.}
    \item{{\tt maMscale}:}{ Object of class "matrix", scale normalization values, rows correspond to spotted probe sequences, columns to arrays in the batch.}
    \item{{\tt maW}:}{ Object of class "matrix", spot quality weights, rows correspond to spotted probe sequences, columns to arrays in the batch.}
    \item{{\tt maLayout}:}{ Object of class "marrayLayout", layout parameters for cDNA microarrays.}
    \item{{\tt maGnames}:}{ Object of class "marrayInfo", description of spotted probe sequences.}
    \item{{\tt maTargets}:}{ Object of class "marrayInfo", description of target samples hybridized to the arrays.}
    \item{{\tt maNotes}:}{ Object of class "character",  any notes concerning the microarray experiments, e.g. hybridization or scanning conditions.}
    \item{{\tt maNormCall}:}{ Object of class "call", function call for normalizing the batch of arrays.}
\end{description}

Most microarray objects contain an {\tt maNotes} slots which may be used to store any string of characters describing the experiments, for examples, notes on the printing, hybridization, or scanning conditions.


\subsection{Creating and accessing slots of microarray objects} 

{\bf Creating new objects.} The function {\tt new} from the {\tt
  methods} package may be used to create new objects from a given
class. For example, to create an object of class {\tt marrayInfo}
describing the target samples in the Swirl experiment, one could use the
following code  

<<eval=TRUE>>=
zebra.RG<-as.data.frame(cbind(c("swirl","WT","swirl","WT"),
c("WT","swirl","WT","swirl")))
dimnames(zebra.RG)[[2]]<-c("Cy3","Cy5")
zebra.samples<-new("marrayInfo",
		    maLabels=paste("Swirl array ",1:4,sep=""), 
		    maInfo=zebra.RG,
		    maNotes="Description of targets for Swirl experiment")
zebra.samples
@

Slots which are not specified in {\tt new} are initialized to the
prototype for the corresponding class. These are usually "empty", e.g.,
{\tt matrix(0,0,0)}. In most cases, microarray objects can be created
automatically using the input functions and their corresponding widgets
in the {\tt marrayInput} package. These were used to create the object
{\tt swirl} of class {\tt marrayRaw}.\\


{\bf Accessing slots.} Different components or slots of the microarray
objects may be accessed using the operator {\tt @}, or alternately, the
function {\tt slot}, which evaluates the slot name. For example, to
access the {\tt maLayout} slot in the object {\tt swirl} and the {\tt
maNgr} slot in the layout object {\tt L}

<<eval=TRUE>>=
L<-slot(swirl, "maLayout")
L@maNgr
@

The function {\tt slotNames} can be used to get information on the slots of a formally defined class or an instance of the class. For example, to get information on the slots for the {\tt marrayLayout} class or on the slots for the object {\tt swirl} use

<<eval=TRUE>>=
slotNames("marrayLayout")
slotNames(swirl)
@

\subsection{Testing the validity of an object}

The function {\tt validObject} from the R package {\tt methods} may be
used to test the validity of an object with respect to its class
definition. This function has two arguments: {\tt object}, the object to
be tested; and {\tt test}. If {\tt test} is TRUE, the function returns a
vector of strings describing the problems, if any.

<<eval=TRUE, echo=TRUE>>=
validObject(maLayout(swirl), test=TRUE)
@

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
\section{Basic microarray methods}\label{smethods}


The following basic methods were defined to facilitate manipulation of microarray data objects. 
To see all methods available for a particular class, e.g., {\tt marrayLayout}, or just the print methods

<<eval=FALSE>>==
showMethods(classes="marrayLayout")
showMethods("show",classes="marrayLayout") 
@


\subsection{Printing methods for microarray objects} 

Since there is usually no need to print out fluorescence intensities for thousands of genes, the {\tt print} method was overloaded for microarray classes by simple report generators. For an overview of the available microarray printing methods, type {\tt  methods ? summary}, or to see all summary methods for the session

<<eval=TRUE>>=
showMethods("summary")
@

For example, summary statistics for an object of class {\tt marrayRaw},
 such as {\tt swirl}, can be obtained by {\tt print(swirl)} or simply {\tt swirl}.

<<eval=TRUE>>=
summary(swirl)
@

\subsection{Subsetting methods for microarray objects} 

In many instances, one is interested in accessing only a subset of
arrays in a batch and/or spots in an array. Subsetting methods {\tt "["}
were defined for this purpose.  For an overview of the available
microarray subsetting methods, type {\tt methods ? "["} or to see all
subsetting methods for the session {\tt showMethods("[")}.  When using
the {\tt "["} operator, the first index refers to spots and the second
to arrays in a batch. Thus, to access the first 100 probe sequences in
the second and third arrays in the batch {\tt swirl} use

<<eval=TRUE>>=
swirl[1:100,2:3]
@


\subsection{Methods for accessing slots of microarray objects} 


A number of simple methods were defined to access slots of the
microarray classes.  Using such methods is more general than using the
{\tt slot} function or {\tt @} operator. In particular, if the class
definitions are changed, any function which uses the {\tt @} operator
will need to be modified. When using a method to access the data in the
slot, only that particular method needs to be modified. Thus, to access
the layout information for the array batch {\tt swirl} one may also use
{\tt maLayout(swirl)}.\\ 

 In addition, various methods were defined to compute basic statistics
 from microarray object slots. For instance, for memory management
 reasons, objects of class {\tt marrayLayout} do not store the spot
 coordinates of each probe. Rather, these can be obtained from the
 dimensions of the grid and spot matrices by applying methods: {\tt
 maGridRow}, {\tt maGridCol}, {\tt maSpotRow}, and {\tt maSpotCol} to
 objects of class {\tt marrayLayout}. Print--tip--group coordinates are
 given by {\tt maPrintTip}. Similar methods were also defined to operate
 directly on objects of class {\tt marrayRaw} and {\tt marrayNorm}. The
 commands below may be used to display the number of spots on the array,
 the dimensions of the grid matrix, and the print--tip--group
 coordinates.

<<eval=TRUE>>=
swirl.layout<-maLayout(swirl)
maNspots(swirl)
maNspots(swirl.layout)
maNgr(swirl)
maNgc(swirl.layout)
maPrintTip(swirl[1:10,3])
@

\subsection{Methods for assigning slots of microarray objects}

A number of methods were defined to replace slots of microarray objects,
 without explicitly using the {\tt @} operator or {\tt slot}
 function. These make use of the {\tt setReplaceMethod} function from
 the R {\tt methods} package. 
 As with the accessor methods just described, the assignment methods are
 named after the slots. For example, to replace the {\tt maNotes} slot
 of {\tt swirl.layout} 

<<eval=TRUE>>=
maNotes(swirl.layout)
maNotes(swirl.layout)<- "New value"
maNotes(swirl.layout)
@

To initialize slots of an empty {\tt marrayLayout} object

<<eval=TRUE>>=
L<-new("marrayLayout")
L
maNgr(L)<-4
@


Similar methods were defined to operate on objects of class {\tt
  marrayInfo}, {\tt marrayRaw} and {\tt marrayNorm}.


\subsection{Methods for coercing microarray objects} 

To facilitate navigation between different classes of microarray
objects, we have defined methods for coercing microarray objects from
one class into another. A list of such methods can be obtained by {\tt
  methods ? coerce}. For example, to coerce an object of class {\tt
  marrayRaw} into an object of class {\tt marrayNorm}: 

<<eval=TRUE>>=
swirl.norm<-as(swirl, "marrayNorm")    
@

It is also possible to convert objects of class {\tt marrayRaw} or {\tt
  marrayNorm} into objects of class {\tt exprSet} (see definition in the
{\tt Biobase} package), see package {\tt convert} package for more
  details.

\begin{verbatim}
> library(convert)
> as(normdata, "exprSet")    
\end{verbatim}


\subsection{Functions for computing layout parameters}

In some cases, plate information is not stored in {\tt marrayLayout}
objects when the data are first read into R. We have defined a function
{\tt maCompPlate} which computes plate indices from the dimensions of
the grid matrix and number of wells in a plate. For example, the Swirl
arrays were printed from 384--well plates, but the plate IDs were not
stored in the {\tt fish.gal} file. To generate plate IDs (arbitrarily
labeled by integers starting with 1) and store these in the {\tt
  maPlate} slot of the {\tt marrayLayout} object use 

<<eval=TRUE>>=
maPlate(swirl)<-maCompPlate(swirl,n=384)
@

Similar functions were defined to generate and manipulate spot
coordinates: {\tt maCompCoord}, {\tt maCompInd}, {\tt maCoord2Ind}, {\tt
maInd2Coord}. The function {\tt maGeneTable} produces a table of spot
coordinates and gene names for objects of class {\tt marrayRaw} and {\tt
marrayNorm}.


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