%% \VignetteIndexEntry{PAA tutorial}

\documentclass{article}

<<style, eval=TRUE, echo=FALSE, results=tex>>=
BiocStyle::latex()
@

\title{Protein Microarray Data Analysis using the \Biocpkg{PAA} Package}
\author{Michael Turewicz}

\begin{document}
\SweaveOpts{concordance=TRUE}

\maketitle

\tableofcontents

%------------------------------------------------------------------------------
%                            Introduction
%------------------------------------------------------------------------------
\newpage
\section{Introduction}
\subsection{General information}
Protein Array Analyzer (\Biocpkg{PAA}) is a package for protein microarray data
analysis (esp., \textit{ProtoArray} data). It imports single color (protein)
microarray data that has been saved in \file{gpr} file format. After pre-
processing (background correction, batch filtering, normalization) univariate
feature pre-selection is performed (e.g., using the "minimum M statistic"
approach - hereinafter referred to as "mMs", \cite{mMs}). Subsequently, a
multivariate feature selection is conducted to discover biomarker candidates.
Therefore, either a frequency-based backwards elimination approach or ensemble
feature selection can be used. \Biocpkg{PAA} provides a complete toolbox of
analysis tools including several different plots for results examination and
evaluation.\\
In this vignette the general workflow of \Biocpkg{PAA} will be outlined by
analyzing an exemplary data set that accompanies this package.

\subsection{Installation}
The recommended way to install \Biocpkg{PAA} is to type the commands described
below in the \R{} console \bioccomment{(note: an active internet connection is
needed)}:
<<install, eval=FALSE, results=hide>>=
# only if you install a Bioconductor package for the first time
source("http://www.bioconductor.org/biocLite.R")
# else
library("BiocInstaller")
biocLite("PAA", dependencies=TRUE)
@
This will install \Biocpkg{PAA} including all dependencies.

Furthermore, \Biocpkg{PAA} has an external dependency that is needed to provide
full functionality. This external dependency is the free \textit{C++} software
package \textit{``Random Jungle''} that can be downloaded from
\url{http://www.randomjungle.de/}. \bioccomment{Note: \Biocpkg{PAA} will be
usable without \textit{Random Jungle}. However, it needs this package for random
jungle recursive feature elimination (\textit{RJ-RFE}) provided by the function
\Rfunction{selectFeatures()}. Please follow the instructions for your OS in the
README file to install \textit{Random Jungle} properly on your machine.}

%------------------------------------------------------------------------------
%                            Loading PAA and importing data
%------------------------------------------------------------------------------
\newpage
\section{Loading \Biocpkg{PAA} and importing data}
After launching \R{}, the first step of the exemplary analysis is to load
\Biocpkg{PAA}.
<<start, results=hide>>=
library(PAA)
@



New microarray data should be imported using the function \Rfunction{loadGPR()}
which is mainly a wrapper to \Biocpkg{limma}'s function
\Rfunction{read.maimages()} featuring optional duplicate aggregation for
\textit{ProtoArray} data. \Biocpkg{PAA} supports the import of files in
\file{gpr} file format. The imported data is stored in an expression list object
(\Rclass{EList}, respectively, \Rclass{EListRaw}, see Bioconductor package
\Biocpkg{limma}). Paths to a targets file and to a folder containing \file{gpr}
files (all \file{gpr} files in this folder that are listed in the targets file
will be read) are mandatory arguments. The folder that can be obtained by the
command \Rcode{system.file("extdata", package = "PAA")} contains an exemplary
targets file that can be used as a template. Below, the first 3 rows of this
targets file are shown.
<<targets, eval=TRUE, results=verbatim>>=
targets <- read.table(file=list.files(system.file("extdata", package="PAA"),
 pattern = "^targets", full.names = TRUE), header=TRUE)
print(targets[1:3,])
@
The columns ``ArrayID'', ``FileName'', and ``Group'' are mandatory. ``Batch'' is
mandatory for microarray data that has been processed in batches. The remaining
three columns as well as custom columns containing further information (e.g.,
clinical data) are optional.



If \Robject{array.type} is set to \Rcode{"ProtoArray"} (default) duplicate spots
will be aggregated. After importing, the object can be saved in a
\file{\*.RData} file for further sessions. In the following code chunk,
\Rfunction{loadGPR()} is demonstrated using a exemplary dummy data set that
comes with PAA and has been created from the real data described below.
<<loadGPR, eval=TRUE, results=hide>>=
gpr <- system.file("extdata", package="PAA")
targets <- list.files(system.file("extdata", package="PAA"),
 pattern = "dummy_targets", full.names=TRUE)
dummy.elist <- loadGPR(gpr.path=gpr, targets.path=targets)
save(dummy.elist, file=paste(gpr, "/DummyData.RData",
   sep=""))
@



\Biocpkg{PAA} comes with an exemplary protein microarray data set.
This 20 Alzheimer's disease serum samples vs. 20 controls data is a subset of a
publicly available \textit{ProtoArray} data set. It can be downloaded from the
repository \textit{``Gene Expression Omnibus''} (\textit{GEO},
\url{http://www.ncbi.nlm.nih.gov/geo/}, record ``GSE29676''). It has been
contributed by \textit{Nagele E et al.} \cite{nagele} (note: Because a data
set stored in \file{gpr} files would be too large to accompany this package the
exemplary data is stored as an \file{\*.RData} file).

In the following code chunk, the \Biocpkg{PAA} installation path (where
exemplary data is located) is localized, the new folder \file{demo\_output}
(where all output of the following analysis will be saved) is created, and the
exemplary data set is loaded (note: exceptionally not via
\Rfunction{loadGPR()}).
<<load, results=hide>>=
cwd <- system.file(package="PAA")
dir.create(paste(cwd, "/demo/demo_output", sep=""))
output.path <- paste(cwd, "/demo/demo_output",  sep="")
load(paste(cwd, "/extdata/Alzheimer.RData", sep=""))
@

%------------------------------------------------------------------------------
%                            Pre-processing
%------------------------------------------------------------------------------
\newpage
\section{Pre-processing}
If the microarrays were manufactured or processed in lots/batches, data analysis
will suffer from batch effects resulting in wrong results. Hence, the
elimination of batch effects is a crucial step of data pre-processing. A simple
method to remove the most obvious batch effects is to find features that are
extremely differential in different batches. In \Biocpkg{PAA} this can be done
for two batches using the function \Rfunction{batchFilter()}. This function
takes an \Rclass{EList} or \Rclass{EListRaw} object and the batch-specific
column name vectors \Robject{lot1} and \Robject{lot2} to find differential
features regarding batches/lots. For this purpose, thresholds for p-values
(Student's t-test) and fold changes can be defined. To visualize the
differential features a volcano plot is drawn. Finally, the differential
features are removed and the remaining data is returned.
\begin{center}
<<batchFilter, results=hide, fig=TRUE>>=
lot1 <- elist$targets[elist$targets$Batch=='Batch1','ArrayID']
lot2 <- elist$targets[elist$targets$Batch=='Batch2','ArrayID']
elist <- batchFilter(elist=elist, lot1=lot1, lot2=lot2, p.thresh=0.001,
fold.thresh=3)
@
\end{center}



For background correction \Biocpkg{limma}'s function
\Rfunction{backgroundCorrect()} can be used:
<<backgroundCorrect, results=hide>>=
library(limma)
elist <- backgroundCorrect(elist, method="normexp",
 normexp.method="saddle")
@



Another important step in pre-processing is normalization. To assist in choosing
an appropriate normalization method for a given data set, \Biocpkg{PAA} provides
two functions: \Rfunction{plotNormMethods()} and \Rfunction{plotMAPlots()}.
\Rfunction{plotNormMethods()} draws boxplots (one boxplot per sample) of raw
data and data after all kinds of normalization provided by \Biocpkg{PAA}. For
each normalization approach sample-wise boxplots are created. All boxplots will
be saved as a high-quality \file{tiff} file, if an output path is specified.
\begin{center}
<<plotNormMethods, eval=FALSE, results=hide, fig=FALSE>>=
plotNormMethods(elist=elist)
@
\end{center}



\Rfunction{plotMAPlots()} draws MA plots of raw data and data after applying all
kinds of normalization methods provided by \Biocpkg{PAA}. If \Rcode{idx="all"}
and an output path is defined (default), for each microarray one \file{tiff}
file containing MA plots will be created. If \Robject{idx} is an integer
indicating the column index of a particular sample, MA plots only for this
sample will be created.
\begin{center}
<<plotMAPlots, results=hide, fig=TRUE>>=
plotMAPlots(elist=elist, idx=10)
@
\end{center}



After choosing a normalization method, the function
\Rfunction{normalizeArrays()} can be used in order to normalize the data.
\Rfunction{normalizeArrays()} takes an \Rclass{EListRaw} object, normalizes the
data, and returns an \Rclass{EList} object containing normalized data in log2
scale. As normalization methods \Rcode{"cyclicloess"}, \Rcode{"quantile"} or
\Rcode{"vsn"} can be chosen. Furthermore, for \textit{ProtoArrays} robust linear
normalization  (\Rcode{"rlm"}, see \textit{Sboner A. et al.} \cite{sboner}) is
provided.
<<normalizeArrays, results=verbatim, fig=FALSE>>=
elist <- normalizeArrays(elist=elist, method="cyclicloess",
cyclicloess.method="fast")
@



In addition to \Rfunction{batchFilter()}, the function \Rfunction{batchAdjust()}
can be used after normalization via \Rfunction{normalizeArrays()} to adjust the
data for batch effects. This is a wrapper to \Biocpkg{sva}'s function
\Rfunction{ComBat()} for batch adjustment using the empirical Bayes approach
\cite{johnson}. To use \Rfunction{batchAdjust()} the targets file information of
the \Rclass{EList} object must contain the columns \textit{``Batch''} and
\textit{``Group''}.
<<batchAdjust, results=verbatim>>=
elist <- batchAdjust(elist=elist, log=TRUE)
@



Since for further analysis also data in original scale will be needed, a copy of
the \Rclass{EList} object containing unlogged data should be created.
<<unlog, results=hide>>=
elist.unlog <- elist
elist.unlog$E <- 2^(elist$E)
@

%------------------------------------------------------------------------------
%                            Differential analysis
%------------------------------------------------------------------------------
\newpage
\section{Differential analysis}
The goal of univariate differential analysis is to detect relevant differential
features. Therefore, statistical measures such as t-test p-values or mMs as well
as fold changes are considered. \Biocpkg{PAA} provides plotting functions in
order to depict the number and the quality of the differential features in the
data set.  Accordingly, the function \Rfunction{volcanoPlot()} draws a volcano
plot to visualize differential features. Therefore, thresholds for p-values and
fold changes can be defined. Furthermore, the p-value computation method
(\Rcode{"mMs"} or \Rcode{"tTest"}) can be set. When an output path is defined
(via \Robject{output.path}) the plot will be saved as a \file{tiff} file. In the
next code chunk, an example with \Rcode{method="tTest"} is given.
\begin{center}
<<volcanoPlot1, eval=TRUE, results=hide, fig=TRUE>>=
c1 <- paste(rep("AD",20), 1:20, sep="")
c2 <- paste(rep("NDC",20), 1:20, sep="")
volcanoPlot(elist=elist.unlog, group1=c1, group2=c2, method="tTest",
p.thresh=0.01, fold.thresh=2)
@
\end{center}
Here, an example with \Rcode{method="mMs"} is given:
<<volcanoPlot2, eval=FALSE, results=hide, fig=FALSE>>=
mMs.matrix1 <- mMs.matrix2 <- mMsMatrix(x=20, y=20)
volcanoPlot(elist=elist.unlog, group1=c1, group2=c2, method="mMs",
p.thresh=0.01, fold.thresh=2, mMs.matrix1=mMs.matrix1,
mMs.matrix2=mMs.matrix2, above=1500, between=400)
@



Another plotting function is \Rfunction{pvaluePlot()} which draws a plot of
p-values for all features in the data set (sorted in increasing order and in
log2 scale). The p-value computation method (\Rcode{"tTest"} or \Rcode{"mMs"})
can be set via the argument \Robject{method}. Furthermore, when
\Rcode{adjust=TRUE} adjusted p-values (method: Benjamini \& Hochberg, 1995,
computed via \Rfunction{p.adjust()}) will be used. For a better orientation,
horizontal dashed lines indicate which p-values are smaller than 0.05 and 0.01.
If \Rcode{adjust=FALSE}, additionally, the respective Bonferroni significance
threshold (to show p-values that would be smaller than 0.05 after a possible
Bonferroni correction) for the given data is indicated by a third dashed line.
\bioccomment{Note: Bonferroni is not used for the adjustment. The dashed line is
for better orientation only.} When an output path is defined (via
\Robject{output.path}) the plot will be saved as a \file{tiff} file. In the next
code chunk, an example with \Rcode{method="tTest"} is given.
\begin{center}
<<pvaluePlot1, eval=TRUE, results=hide, fig=TRUE>>=
pvaluePlot(elist=elist.unlog, group1=c1, group2=c2, method="tTest")
@
\end{center}
Here, an example with \Rcode{method="mMs"} is given:
<<pvaluePlot2, eval=FALSE, results=hide, fig=FALSE>>=
mMs.matrix1 <- mMs.matrix2 <- mMsMatrix(x=20, y=20)
pvaluePlot(elist=elist.unlog, group1=c1, group2=c2, method="mMs",
mMs.matrix1=mMs.matrix1, mMs.matrix2=mMs.matrix2, above=1500,
between=400)
@
Here, an example with \Rcode{method="tTest"} and \Rcode{adjust=TRUE} is given:
\begin{center}
<<pvaluePlot3, eval=TRUE, results=hide, fig=TRUE>>=
pvaluePlot(elist=elist.unlog, group1=c1, group2=c2, method="tTest", adjust=TRUE)
@
\end{center}
Here, an example with \Rcode{method="mMs"} and \Rcode{adjust=TRUE} is given:
<<pvaluePlot4, eval=FALSE, results=hide, fig=FALSE>>=
mMs.matrix1 <- mMs.matrix2 <- mMsMatrix(x=20, y=20)
pvaluePlot(elist=elist.unlog, group1=c1, group2=c2, method="mMs",
mMs.matrix1=mMs.matrix1, mMs.matrix2=mMs.matrix2, above=1500,
between=400, adjust=TRUE)
@



Finally, \Rfunction{diffAnalysis()} performs a detailed univariate differential
analysis. This function takes an \Robject{EList\$E}- or \Robject{EListRaw\$E}-
matrix (e.g., \Rcode{temp <- elist\$E}) extended by row names comprising
\textit{``BRC''}-IDs of the corresponding features. The BRC-IDs can be
created via:\\
\Rcode{brc <- paste(elist\$genes[,1], elist\$genes[,3], elist\$genes[,2])}.\\
Next, the row names can be assigned as follows: \Rcode{rownames(temp) <- brc}.
Furthermore, the corresponding column name vectors, group labels and mMs-
parameters are needed to perform the univariate differential analysis. This
analysis covers inter alia p-value computation, p-value adjustment (method:
Benjamini \& Hochberg, 1995), and fold change computation. Since the results
table is usually large, a path for saving the results should be defined via
\Robject{output.path}. Optionally, a vector of row indices (\Robject{features})
and additionally (not mandatory for subset analysis) a vector of corresponding
feature names (\Robject{feature.names}) can be forwarded to perform the analysis
for a feature subset.
<<diffAnalysis, eval=TRUE, results=verbatim, fig=FALSE>>=
E <- elist.unlog$E
rownames(E) <- paste(elist.unlog$genes[,1], elist.unlog$genes[,3],
    elist.unlog$genes[,2])
write.table(x=cbind(rownames(E),E), file=paste(cwd,"/demo/demo_output/data.txt",
    sep=""), sep="\t", eol="\n", row.names=FALSE, quote=FALSE)
mMs.matrix1 <- mMs.matrix2 <- mMsMatrix(x=20, y=20)
diff.analysis.results <- diffAnalysis(input=E, label1=c1, label2=c2,
    class1="AD", class2="NDC", output.path=output.path,
    mMs.matrix1=mMs.matrix1, mMs.matrix2=mMs.matrix2, above=1500,
    between=400)
print(diff.analysis.results[1:10,])
@
Subsequently, the most relevant differential features (i.e., features having low
p-values and high absolute fold changes) can be extracted as a univariate
feature selection. Nevertheless, it is recommended to perform also multivariate
feature selection and to consider feature panels obtained from both approaches.

%------------------------------------------------------------------------------
%                            Feature pre-selection
%------------------------------------------------------------------------------
\newpage
\section{Feature pre-selection}
Before multivariate feature selection will be performed, it is recommended to
discard features that are obviously not differential. Discarding them will
accelerate runtimes without any negative impact on results. In \Biocpkg{PAA},
this task is called \textit{``feature pre-selection''} and it is performed by
the function \Rfunction{preselect()}. This function iterates all features of the
data set to score them via \textit{mMs}, \textit{Student's t-test}, or
\textit{mRMR}. If \Robject{discard.features} is \Rcode{TRUE} (default), all
features that are considered as obviously not differential will be collected and
returned for discarding. Which features are considered as not differential
depends on the parameters \Robject{method}, \Robject{discard.threshold}, and
\Robject{fold.thresh}.\\
\begin{itemize}
 \item If \Rcode{method = "mMs"}, features having an \textit{mMs} value larger
 than \Robject{discard.threshold} (here: numeric between 0.0 and 1.0) or do not
 satisfy the minimal absolute fold change \Robject{fold.thresh} will be
 considered as not differential.\\
 \item If \Rcode{method = "tTest"}, features having a p-value larger than
 \Robject{discard.threshold} (here: numeric between 0.0 and 1.0) or do not
 satisfy the minimal absolute fold change \Robject{fold.thresh} will be
 considered as not differential.\\
 \item If \Rcode{method = "mrmr"}, \textit{mRMR} scores for all features will be
 computed as scoring method (using the function \Rfunction{mRMR.classic()} of
 the \R{} package \CRANpkg{mRMRe}). Subsequently, features that are not the
 \Robject{discard.threshold} (here: integer indicating a number of features)
 features having the best \textit{mRMR} scores are considered as not
 differential.
\end{itemize}
<<preselect, eval=TRUE, results=verbatim, fig=FALSE>>=
mMs.matrix1 <- mMs.matrix2 <- mMsMatrix(x=20, y=20)
pre.sel.results <- preselect(elist=elist.unlog, columns1=c1, columns2=c2,
    label1="AD", label2="NDC", discard.threshold=0.5, fold.thresh=1.5,
    discard.features=TRUE, mMs.above=1500, mMs.between=400,
    mMs.matrix1=mMs.matrix1, mMs.matrix2=mMs.matrix2,
    method="mMs")
elist <- elist[-pre.sel.results$discard,]
@

%------------------------------------------------------------------------------
%                            Feature selection
%------------------------------------------------------------------------------
\newpage
\section{Feature selection}
For multivariate feature selection \Biocpkg{PAA} provides the function
\Rfunction{selectFeatures()}. It performs a multivariate feature selection using
\textit{``frequency-based''} feature selection (based on \textit{RF-RFE},
\textit{RJ-RFE} or \textit{SVM-RFE}) or \textit{``ensemble''} feature selection
(based on \textit{SVM-RFE}).

\textbf{Frequency-based feature selection (\Rcode{method="frequency"}):} The
whole data is splitted in k cross validation training and test set pairs. For
each training set a multivariate feature selection procedure is performed. The
resulting k feature subsets are tested using the corresponding test sets (via
classification). As a result, \Rfunction{selectFeatures()} returns the average
k-fold cross validation classification accuracy as well as the selected feature
panel (i.e., the union set of the k particular feature subsets). As multivariate
feature selection methods random forest recursive feature elimination
(\textit{RF-RFE}), random jungle recursive feature elimination (\textit{RJ-RFE})
and support vector machine recursive feature elimination (\textit{SVM-RFE}) are
supported. To reduce running times, optionally, an additional univariate feature
pre-selection can be performed (usage via \Robject{preselection.method}). As
univariate pre-selection methods mMs (\Rcode{"mMs"}), Student's t-test
(\Rcode{"tTest"}) and mRMR (\Rcode{"mrmr"}) are supported. Alternatively, no
pre-selection can be chosen (\Rcode{"none"}). This approach is similar to the
method proposed in \textit{Baek et al.} \cite{baek}.

\textbf{Ensemble feature selection (\Rcode{method="ensemble"}):} From the whole
data a previously defined number of subsamples is drawn defining pairs of
training and test sets. Moreover, for each training set a previously defined
number of bootstrap samples is drawn. Then, for each bootstrap sample
\textit{SVM-RFE} is performed and a feature ranking is obtained. To obtain a
final ranking for a particular training set, all associated bootstrap rankings
are aggregated to a single ranking. To score the \Robject{cutoff} best features,
for each subsample a classification of the test set is performed (using a svm
trained with the \Robject{cutoff} best features from the training set) and the
classification accuracy is determined. Finally, the stability of the
subsample-specific panels is assessed (via Kuncheva index,
\textit{Kuncheva LI, 2007} \cite{kuncheva}), all subsample-specific rankings are
aggregated, the top n features (defined by \Robject{cutoff}) are selected, the
average classiification accuracy is computed, and all these results are
returned in a list. This approach has been proposed and is described in
\textit{Abeel et al.} \cite{abeel}.

\Rfunction{selectFeatures()} takes an \Robject{EListRaw} or \Robject{EList}
object, group-specific sample numbers, group labels and parameters choosing and
setting up a univariate feature pre-selection method as well as a multivariate
feature selection method (frequency-based or ensemble feature selection) to
select a panel of differential features. When an output path is defined (via
\Robject{output.path}) results will be saved on the hard disk and when
\Robject{verbose} is TRUE additional information will be printed to the console.
Depending on the selection method, one of two different results lists will be
returned:\\

\begin{enumerate}
\item If \Robject{method} is \Rcode{"frequency"}, the results list contains the
following elements:
\begin{itemize}
  \item accuracy: average k-fold cross validation accuracy.
  \item sensitivity: average k-fold cross validation sensitivity.
  \item specificity: average k-fold cross validation specificity.
  \item features: selected feature panel.
  \item all.results: complete cross validation results.
\end{itemize}

\item If \Robject{method} is \Rcode{"ensemble"}, the results list contains the
following elements:
\begin{itemize}
  \item accuracy: average accuracy regarding all subsamples.
  \item sensitivity: average sensitivity regarding all subsamples.
  \item specificity: average specificity regarding all subsamples.
  \item features: selected feature panel.
  \item all.results: all feature ranking results.
  \item stability: stability of the feature panel (i.e., Kuncheva index for the
  subrun-specific panels).
\end{itemize}
\end{enumerate}
\newpage
In the following two code chunks first \textit{``frequency-based''} feature
selection and then \textit{``ensemble''} feature selection is demonstrated.
<<selectFeatures1, eval=FALSE, results=hide>>=
selectFeatures.results <- selectFeatures(elist,n1=20,n2=20,label1="AD",
    label2="NDC",selection.method="rf.rfe",subruns=2,candidate.number=1000,
    method="frequency")
@
<<selectFeatures2, eval=FALSE, results=hide>>=
selectFeatures.results <- selectFeatures(elist,n1=20,n2=20,label1="AD",
    label2="NDC",selection.method="rf.rfe",subsamples=10,bootstraps=10,
    method="ensemble")
@
Because runtimes would take too long for this vignette \Biocpkg{PAA} comes with
pre-computated \Robject{selectFeatures.results} objects stored in
\file{\*.RData} files. These objects can be loaded as follows:
<<loadSelectFeaturesResults, eval=TRUE, results=hide>>=
# results of frequency-based feature selection:
load(paste(cwd, "/extdata/selectFeaturesResultsFreq.RData", sep=""))
# or results of ensemble feature selection:
load(paste(cwd, "/extdata/selectFeaturesResultsEns.RData", sep=""))
@

%------------------------------------------------------------------------------
%                            Results inspection
%------------------------------------------------------------------------------
\newpage
\section{Results inspection}
After the selection of a feature panel, these features should be validated by
manual inspection and evaluation for further research. To aid results
inspection, \Biocpkg{PAA} provides several functions. The function
\Rfunction{plotFeatures()} plots the intensities of all features (represented by
BRC-IDs) that have been selected by \Rfunction{selectFeatures()} (one sub-plot
per feature) in group-specific colors. All sub-plots are aggregated in one
figure. If \Robject{output.path} is not NULL, this figure will be saved in a
\file{tiff} file in \Robject{output.path}.
\begin{center}
<<plotFeatures, eval=TRUE, results=verbatim, fig=TRUE>>=
plotFeatures(features=selectFeatures.results$features, elist=elist, n1=20,
    n2=20, group1="AD", group2="NDC")
@
\end{center}

Alternatively, the function \Rfunction{plotFeaturesHeatmap()} plots intensities
of all features given in the vector \Robject{features} (represented by BRC-IDs)
as a heatmap. If \Robject{description} is \Rcode{TRUE} (default:
\Rcode{FALSE}), features will be described via protein names instead of
uniprot accessions. Again, if \Robject{output.path} is not \Rcode{NULL}, the
heatmap will be saved as a \file{tiff} file in \Robject{output.path}.
\begin{center}
<<plotFeaturesHeatmap, eval=TRUE, results=verbatim, fig=TRUE>>=
plotFeaturesHeatmap(features=selectFeatures.results$features, elist=elist,
    n1=20, n2=20, description=TRUE)
@
\end{center}

Finally, the function \Rfunction{printFeatures()} creates a table containing the
selected biomarker candidate panel as well as additional information for results
inspection. If \Robject{output.path} is defined, this table will be saved in a
\file{txt} file (\file{candidates.txt}).
\begin{center}
<<printFeatures, eval=TRUE, results=verbatim, fig=FALSE>>=
printFeatures(features=selectFeatures.results$features, elist=elist.unlog)[,-2]
@
\end{center}

%------------------------------------------------------------------------------
%                            References
%------------------------------------------------------------------------------
\begin{thebibliography}{9}
\bibitem{mMs}
Love B: The Analysis of Protein Arrays. In: Functional Protein Microarrays in
Drug Discovery. CRC Press; 2007: 381-402.

\bibitem{nagele}
Nagele E, Han M, Demarshall C, Belinka B, Nagele R (2011): Diagnosis of
Alzheimer's disease based on disease-specific autoantibody profiles in human
sera. PLoS One 6: e23112.

\bibitem{sboner}
Sboner A. et al., Robust-linear-model normalization to reduce technical
variability in functional protein microarrays. J Proteome Res 2009,
8(12):5451-5464.

\bibitem{johnson}
Johnson WE, Li C, and Rabinovic A (2007) Adjusting batch effects in microarray
expression data using empirical Bayes methods. Biostatistics 8:118-27.

\bibitem{baek}
Baek S, Tsai CA, Chen JJ.: Development of biomarker classifiers from high-
dimensional data. Brief Bioinform. 2009 Sep;10(5):537-46.

\bibitem{kuncheva}
Kuncheva, LI: A stability index for feature selection. Proceedings of the IASTED
International Conference on Artificial Intelligence and Applications.
February 12-14, 2007. Pages: 390-395.

\bibitem{abeel}
Abeel T, Helleputte T, Van de Peer Y, Dupont P, Saeys Y: Robust biomarker
identification for cancer diagnosis with ensemble feature selection methods.
Bioinformatics. 2010 Feb 1;26(3):392-8.

\end{thebibliography}

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