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<h1 class="documentFirstHeading">Lab4b.Rnw</h1>
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<p>%
% NOTE -- ONLY EDIT THE .Rnw FILE!!! The .tex file is
% likely to be overwritten.
%
% \VignetteIndexEntry{Seattle Lab 4B}
%\VignetteDepends{Biobase,affy}
%\VignetteKeywords{Microarray}
\documentclass<a href="#ref12pt">[12pt]</a>{article}</p>
<p>\usepackage{amsmath,pstricks}
\usepackage<a href="#refauthoryear,round">[authoryear,round]</a>{natbib}
\usepackage{hyperref}</p>
<p>\textwidth=6.2in
\textheight=8.5in
%\parskip=.3cm
\oddsidemargin=.1in
\evensidemargin=.1in
\headheight=-.3in</p>
<p>\newcommand{\scscst}{\scriptscriptstyle}
\newcommand{\scst}{\scriptstyle}</p>
<p>\bibliographystyle{plainnat}</p>
<p>\title{Lab 4B: An Introduction to Bioconductor's {\tt affy} package}</p>
<p>\begin{document}</p>
<p>\maketitle</p>
<p>In this lab, we demonstrate the main functions in the \verb+affy+
package for pre-processing Affymetrix microarray data. To load the
package</p>
<p><<loadpacks>>=
library(affy)
@</p>
<p>For a more detailed introduction, consult the package vignettes which can be listed
by the command {\tt openVignette("affy")}. A demo can also be accessed
by {\tt demo(affy)}. A number of sample datasets are available in the
package; to list these, type {\tt data(package="affy")}.</p>
<p>We will work mainly with the \verb+Dilution+ dataset.
For a description of \verb+Dilution+, type {\tt ? Dilution}.
To load this dataset </p>
<p><<loaddata>>=
data(Dilution)
@</p>
<p>%%%%%%%%%%%%%%%%%%%%%%%%%
%%% affy classes</p>
<p>One of the main classes in \verb+affy+ is the \verb+AffyBatch+
class. For details on this class consult the help file, {\tt ?
AffyBatch}; methods for manipulating instances of this class are also
described in the help file. Other classes include \verb+ProbeSet+ (PM and MM
intensities for individual probe sets), \verb+Cdf+ (information
contained in a CDF file), and
\verb+Cel+ (single array cel intensity data).
The object \verb+Dilution+ is an instance of the class
\verb+AffyBatch+. Try the following commands to obtain information of
this object</p>
<p><<dil>>=
class(Dilution)
slotNames(Dilution)
Dilution
annotation(Dilution)
@</p>
<p>For a description of the target samples hybridized to the arrays
<<pheno>>=
phenoData(Dilution)
pData(Dilution)
@</p>
<p>The \verb+exprs+ slot contains a matrix with columns corresponding to
arrays and rows to individual probes on the array.
To obtain the matrix of intensities for all four arrays
<<expr>>=
e<-exprs(Dilution)
nrow(Dilution)*ncol(Dilution)
dim(e)
@</p>
<p>You can access probe-level PM and MM intensities using
<<pmmm>>=
PM<-pm(Dilution)
dim(PM)
PM<a href="#ref1:5,">[1:5,]</a>
@</p>
<p>To get the probe set names (Affy IDs)</p>
<p><<AffyID>>=
gnames<-geneNames(Dilution)
length(gnames)
gnames<a href="#ref1:5">[1:5]</a>
nrow(e)/length(gnames)
@</p>
<p>As with other microarray objects in Bioconductor packages, you can
use subsetting commands for {\tt AffyBatch} objects</p>
<p><<sub>>=
dil1<-Dilution<a href="#ref1">[1]</a>
class(dil1)
dil1
cel1<-Dilution[<a href="#ref1">[1]</a>]
class(cel1)
cel1
@</p>
<p>%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% Reading in data</p>
<p>One of the main function for reading in Affymetrix data is
\verb+ReadAffy+. It reads in data from \verb+CEL+ and \verb+CDF+
files and creates objects of class \verb+AffyBatch+. Using
\verb+ReadAffy(widget=TRUE)+ provides widgets for interactive data input.</p>
<p>%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% Diagnostic plots</p>
<p>To produce a spatial image of probe log intensities and probe raw intensities
<<image1,fig=TRUE>>=
# Log transformation
image(Dilution<a href="#ref1">[1]</a>)
@</p>
<p><<image2,fig=TRUE>>=
# No transformation
image(cel1)
@</p>
<p>To produce boxplots of probe log intensities
<<boxplot,fig=TRUE>>=
boxplot(Dilution,col=c(2,2,3,3))
@
Note that scanner effects seem stronger than concentration effect. </p>
<p>To produce density plots of probe log intensities
<<hist,fig=TRUE>>=
hist(Dilution, type="l", col=c(2,2,3,3), lty=rep(1:2,2), lwd=3)
@</p>
<p>%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% Normalization</p>
<p>The \verb+affy+ package provides implementations for a number of methods
for background estimation, probe-level normalization (e.g., quantile,
curve-fitting (Bolstad et al., 2002)), and computation
of expression measures (e.g., MAS 4.0, MAS 5.0, MBEI (Li \& Wong,
2001), RMA (Irizarry et al., 2003)). To list available methods for
\verb+AffyBatch+ objects </p>
<p><<methods>>=
bgcorrect.methods
normalize.AffyBatch.methods
pmcorrect.methods
express.summary.stat.methods
@</p>
<p>The main normalization function is \verb+expresso+. You can select
pre-processing methods interactively using widgets by typing {\tt
expresso(Dilution, widget=TRUE)}. The function operates on objects of class
\verb+AffyBatch+ and returns objects of class \verb+exprSet+.</p>
<p>\verb+rma+ provides a more efficient implementation of Robust Multi-array
Average (RMA)</p>
<p><<rma>>=
rmaDil<-rma(Dilution)
class(rmaDil)
@</p>
<p>%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% CDF data packages</p>
<p>Data packages for CDF information can be download from
\url{www.bioconductor.org}|. These packages contain environment objects
which provide mappings between AffyIDs and matrices of probe
locations, with rows corresponding to probe-pairs and columns to PM and
MM cels. CDF environments for HGU95Av2 and HGU133A chips are
already in the package. For information on the environment object {\tt ? hgu95av2cdf}</p>
<p><<cdf>>=
annotation(Dilution)
data(hgu95av2cdf)
pnames<-ls(env=hgu95av2cdf)
length(gnames)
gnames<a href="#ref1:5">[1:5]</a>
get(gnames<a href="#ref1">[1]</a>,env=hgu95av2cdf)
@</p>
<p>You can also use the \verb+indexProbe+, \verb+pmindex+, and
\verb+mmindex+ to get information on probe location</p>
<p><<loc>>=
plocs<-indexProbes(Dilution,which="both")
plocs[<a href="#ref1">[1]</a>]
pmindex(Dilution,genenames=gnames<a href="#ref1">[1]</a>, xy=TRUE)
pmindex(Dilution,genenames=gnames<a href="#ref1">[1]</a>)
@</p>
<p>\end{document}
</p>
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<a href="http://bioconductor.fhcrc.org/News/AdvancedR"
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Advanced R Programming for Bioinformatics course material now <a href="http://bioconductor.fhcrc.org/workshops/2010/AdvancedR/">available</a>
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Bioconductor 2.6, consisting of 389 packages and designed to work with R version 2.11, was released today.
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