%\VignetteIndexEntry{Supplement: two-way assays}
%\VignetteKeywords{Two-way assays, cell based assays}
%\VignettePackage{cellHTS}

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pdftitle={Analysis of two-way cell-based assays},%
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\begin{document}

%------------------------------------------------------------
\title{Analysis of two-way cell-based assays}
%------------------------------------------------------------
\author{L\'igia Br\'as, Michael Boutros and Wolfgang Huber}
\maketitle
\tableofcontents

\section{Introduction}
This technical report is a supplement of the main vignette 
\textit{End-to-end analysis of cell-based screens: from raw intensity readings
to the annotated hit list} that is given as part of the \Rpackage{cellHTS} 
package, and that accompanies the paper \textit{Analysis
of cell-based RNAi screens} by Michael Boutros, L\'igia Br\'as and
Wolfgang Huber~\cite{Boutros2006}. 

The report explains how the 
\Rpackage{cellHTS} package can be used to document and analyse two-way assays.
We call \emph{two-way assays} to the assays where we can have two types of effectors: 
\emph{activators} and \emph{inhibitors}. In such cases, both type of effectors are used as positive controls, and they need to be considering indivually by the \Rpackage{cellHTS} package.

This text has been produced as a reproducible
document~\cite{Gentleman2004RepRes}, containing the actual computer
instructions, given in the language R, to produce all results, including the figures and 
tables that are depicted here. To reproduce the computations shown here, 
you will need an installation of R (version 2.3 or greater) 
together with a recent version of the package
\Rpackage{cellHTS} and of some other add-on packages. Then, you can simply take the file
\textit{twoWay.Rnw} in the \textit{doc} directory of the package,
open it in a text editor, run it using the R command
\Rfunction{Sweave}, and modify it according to your needs.

We start by loading the package.
%
<<setup1, results=hide>>=
library("cellHTS")
@ 
%
<<setup2, echo=FALSE, results=hide>>=
## for debugging:
options(error=recover)
@ 


%
%------------------------------------------------------------
\section{Assembling the data}
\label{sec:assemble}
%------------------------------------------------------------

The example data set corresponds to one 96-well plate of a two-way assay 
performed with human cells. The screen was performed in duplicate, 
and a luminescence-reporter was used in the assay.


%------------------------------------------------------------
\subsection{Reading the raw intensity files}
%------------------------------------------------------------

The set of available result files and the information about them (which plate,
which replicate) is given in the \emph{plate list file}. 
The first line of the plate list file for this data set is shown in 
Table~\ref{tab:platelist}.
\input{twoWay-platelist}

The path for the raw data is defined below:
%
<<dataPath>>=
experimentName = "TwoWayAssay"
dataPath=system.file(experimentName, package="cellHTS") 
@ 
%
The input files are in the
\Robject{\Sexpr{experimentName}} directory of the \Rpackage{cellHTS}
package. 

The function \Rfunction{readPlateData} of \Rpackage{cellHTS} package reads the plate list file, 
and the intensity files, assembling the data into a single R object.
In this example, the raw intensity files correspond to csv files, in which the first part 
consists of data from the acquisition instrument, while the relevant data values are in the $12 \times 8$ 
matrix at the end of the file. So, we need to give as argument to  
\Rfunction{readPlateData}, a function suitable to import such data file format. 
This function, which we named \Rfunction{importData}, is given in the same directory as the data files, in a R file with the same name, and needs to be sourced:
%
<<source import function>>=
source(file.path(dataPath, "importData.R"))
@ 
%
Finally, we call \Rfunction{readPlateData}:
<<readPlateData, results=hide>>=
x <- readPlateData("Platelist.txt", importFun=importData, name=experimentName, path=dataPath)
@ 
%
<<showX>>=
x
@ 
%
%% Create the tables with the first lines of the plate list file:
%% it would have been nice to use the "xtable" package but it insists on 
%%   adding row numbers, which we don't like.
<<plateFileTable, results=hide, echo=FALSE>>=
cellHTS:::tableOutput(file.path(dataPath, "Platelist.txt"), selRows=1, 
  "plate list", preName="twoWay")
@ 

%------------------------------------------------------------
\subsection{Configuring and annotating the \Rpackage{cellHTS} object}
%------------------------------------------------------------
\input{twoWay-plateconfiguration} 
\input{twoWay-geneID} 

The \emph{plate configuration file} gives the information about the 
content of the plate wells, which is used by the software to annotate the 
measured data. The first lines of this file are shown in 
Table~\ref{tab:plateconfiguration}.
For the sample data, there is no \emph{screen log file}, meaning that no measurements
were marked to be flagged during the assay.
Another file necessary for the configuration step of the \Robject{cellHTS} object is the 
\emph{screen description file}, which gives a general description of the screen.
%
<<configure the data>>=
confFile <- file.path(dataPath, "Plateconf.txt")
descripFile  <- file.path(dataPath, "Description.txt")
x <- configure(x, confFile=confFile, descripFile = descripFile)
@ 
%
In this data set, instead of using the default names \emph{pos} and \emph{neg} for 
positive and negative controls, respectively, we have used the names of the target genes.
Thus, the well annotation in this sample plate looks like this~\footnote{Note that when 
reading the plate configuration file, the \Rfunction{configure} function 
puts the content of the column \emph{Content} 
in lowercase. However, the orignal content of this column 
(and the complete plate configuration file) is stored in \Robject{x\$plateConf}, 
so if you prefer, you can use the command \Robject{table(x\$plateConf\$Content)} 
instead of \Robject{table(x\$wellAnno)}.}:
%
<<well annottaion>>=
table(x$wellAnno)
@ 
Here, \emph{AATK}, \emph{ATTK} and \emph{MAP2K6} are positive controls, whereas
\emph{GFP} and \emph{mock} (mock transfection) correspond to negative controls. 
We will return to this issue in Section~\ref{sec:preprocess}, but now, we will proceed to 
the next step in the data assemble stage, whereby we add the annotation information 
to \Robject{x}. This information is in the \emph{screen annotation file}, and 
Table~\ref{tab:geneID} shows the first 5 lines of this file.
Besides the mandatory columns \emph{Plate}, \emph{Well} and \emph{GeneID}, 
the file contains the optional column \emph{GeneSymbol}, whose content will be used for
the tooltips display in the HTML quality reports that will be produced later on.
%
<<annotate the data>>=
geneIDFile <- file.path(dataPath, "GeneIDs.txt")
x <- annotate(x, geneIDFile)
@ 
%
% Create the table for plateConf
<<plateConfscreenLogTable, results=hide, echo=FALSE>>=
cellHTS:::tableOutput(confFile, "plate configuration", selRows=1:4, 
  preName="twoWay")
@ 
%% Create the table for screen annotation:
<<geneIDsTable, results=hide, echo=FALSE>>=
cellHTS:::tableOutput(geneIDFile, "gene ID", selRows = 3:6, preName="twoWay")
@ 


%------------------------------------------------------------
\section{Data preprocessing and summarization of replicates}
\label{sec:preprocess}
%------------------------------------------------------------ 

We can take a first look at the data by constructing the HTML quality reports 
using the \Rfunction{writeReport} function. 
For that, we must define the positive and negative controls needed when calling 
this function.\\

For a 2-way assay, the argument \Robject{posControls} of 
\Rfunction{writeReport} should be defined as a list with two components: 
\emph{act} and \emph{inh}, which should be defined as vectors of regular 
expressions with the same length as the number of channels (in this case, length one).
These arguments will be passed to the \Rfunction{regexpr} function 
for pattern matching within the well annotation given in \Robject{x\$wellAnno}.\\

In the example presented here, in the positive controls, we have 
\emph{AATK} and \emph{ATTK} as activator effectors, and \emph{MAP2K6} as 
an inhibitor effector.
The negative controls are \emph{GFP} and the mock transfection 
(indicated as \emph{mock} in the plate configuration file).
Thus, we define the negative and positive controls as follows:
%
<<define controls>>=
negCtr <- "(?i)^GFP$|^mock$"
posCtr <- list(act = "(?i)^AATK$|^ATTK$", inh = "(?i)^MAP2K6$")
@ 
%
Finally, we construct the quality report pages for the raw data in a directory called \Robject{2Wraw}, in the working directory:
%
<<writeReport1Show, eval=FALSE>>=
out <- writeReport(x, outdir="2Wraw",
     posControls=posCtr, negControls=negCtr, 
     plotPlateArgs=list(xrange=c(300, 4000)))
@ 
%
<<writeReport1Do, echo=FALSE, results=hide>>=
out <- writeReport(x, force=TRUE, outdir="2Wraw", 
   posControls=posCtr, negControls=negCtr, 
   plotPlateArgs=list(xrange=c(300, 4000)))
@ 
%
After this function has finished, we can view the index page of
the report:
%
<<browseReport1, eval=FALSE>>=
browseURL(out)
@ 
%
As can be seen in the HTML reports, the dynamic range for each replicate (and the respective average) 
was calculated separately for the activators and inhibitors.\\

Next, we normalize the data using a simple approach.
For each replicate, the plate intensity values at the negative control wells are taken 
as a correction factor, so that each plate measurement is divided by it. 
Then, the obtained values are $\log_2$ transformed.
%
<<normalization>>=
x <- normalizePlates(x, normalizationMethod ="negatives", 
   negControls = negCtr, transform=log2)
@ 
%
The normalized intensities are stored in the slot
\Robject{x\$xnorm}, in an array of the same size as
\Robject{x\$xraw}.

Below, we call the function \Rfunction{summarizeReplicates} to determine the 
$z$-score values for each replicate, and then summarize 
the replicated $z$-score values by taking the average. A more conservative approach would be to consider the $z$-score value closest to zero between replicates (\Robject{summary}='closestToZero').
%
<<summarizeReplicates>>=
x <- summarizeReplicates(x, zscore="+", summary="mean") 
@ 
%
The resulting single $z$-score value per probe is stored in the
slot \Robject{x\$score}. Figure~\ref{twoWay-boxplotzscore} shows the boxplots of 
the $z$-scores for the different types of probes.
%
<<boxplotzscore, fig=TRUE, include=FALSE, width=7, height=6>>=
ylim <- quantile(x$score, c(0.001, 0.999), na.rm=TRUE)
boxplot(x$score ~ x$wellAnno, col="lightblue", main="scores", outline=FALSE, ylim=ylim, xaxt="n")
lab <- unique(x$plateConf$Content)
lab <- lab[match(levels(x$wellAnno), tolower(lab))]
axis(1, at=c(1:nlevels(x$wellAnno)), labels=lab)
@ 
%
\myincfig{twoWay-boxplotzscore}{0.8\textwidth}{Boxplots of $z$-scores 
for the different types of probes.}
%
Now that the data have been preprocessed and scored, we call again 
\Rfunction{writeReport} and use a web browser to view the resulting report:
%

<<report2Show, eval=FALSE, results=hide>>=
out <- writeReport(x, outdir="2Wnormalized", posControls=posCtr, negControls=negCtr, 
   plotPlateArgs=list(xrange=c(-1,1)), imageScreenArgs=list(zrange=c(-2,3)))
@
<<report2Do, results=hide, echo=FALSE, results=hide>>=
out <- writeReport(x, outdir="2Wnormalized", posControls=posCtr, negControls=negCtr, 
   plotPlateArgs=list(xrange=c(-1,1)), imageScreenArgs=list(zrange=c(-2,3)), force=TRUE)
@
<<browse2, eval=FALSE>>=
browseURL(out)
@ 
%
The quality reports have been created in the folder \Robject{2Wnormalized} 
in the working directory. Now the report shows also controls-related plots, 
distinguishing the two types of positive controls (activators and inhibitors).
Furthermore, the report shows the image plot with the final scored values 
for the whole data set.
Finally, we save the data set to a file. 
%
<<savex>>=
save(x, file=paste(experimentName, ".rda", sep=""))
@ 
% 

%------------------------------------------------------------
%Bibliography
%------------------------------------------------------------
\bibliography{cellhts}
\bibliographystyle{plain}

\end{document}


