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%\VignetteKeywords{copy number analysis, mixture distribution, latent variables, Poisson distribution,
% EM algorithm, NGS, CNV, copy number variant}


\documentclass[article]{bioinf}

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   pdftitle={cn.mops - Mixture of Poissons for CNV detection in NGS data},
   pdfauthor={G\"unter Klambauer}}

\title{cn.mops - Mixture of Poissons for CNV detection in NGS data}
\author{G\"unter Klambauer}
\affiliation{Institute of Bioinformatics, Johannes Kepler University
Linz\\Altenberger Str. 69, 4040 Linz, Austria\\
\email{cn.mops@bioinf.jku.at}}

\newcommand{\cnmops}{\texttt{cn.mops}}
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\begin{document}
<<echo=FALSE>>=
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set.seed(0)
library(cn.mops)
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@
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%\section*{Scope and Purpose of this Document}
%
%This document is a user manual for the \R\ package \cnmops.
%It is only meant as a gentle introduction into how to use the basic
%functions implemented in this package. Not all features of the \R\
%package are described in full detail. Such details can be obtained
%from the documentation enclosed in the  \R\ package. Further note
%the following: (1) this is neither an introduction to CNV detection from NGS 
%data; (2) this is not an introduction to \R.
%If you lack the background for understanding this manual, you first
%have to read introductory literature on these subjects.
%


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\section{Introduction}
The \cnmops\ package is part of the Bioconductor 
(\url{http://www.bioconductor.org}) project. The package allows to 
detect copy number variations (CNVs) from next generation sequencing (NGS) 
data sets  based on a generative model. Please visit
\url{http://www.bioinf.jku.at/software/cnmops/cnmops.html} for additional
information.\par 

To avoid the false discoveries induced by read count variations along
the chromosome or across samples, we
propose a ``Mixture Of PoissonS model for CNV detection'' (\method{cn.mops}). 
The \method{cn.mops} model is not affected by read count variations along the chromosome, because
at each DNA position a local model is constructed. 
Read count variations across samples are decomposed by the
\method{cn.mops} model
into integer copy numbers and noise by
its mixture components and Poisson distributions, respectively.
In contrast to existing methods, \method{cn.mops} model's posterior provides integer
copy numbers together with their uncertainty. Model selection in a
Bayesian framework is based on maximizing the posterior given the
samples by an expectation
maximization (EM) algorithm. 
The model incorporates
the linear dependency between average read counts in a DNA segment and
its copy number. 
Most importantly, a Dirichlet prior on the mixture
components prefers constant copy number 2 for all samples. 
The more the data drives the posterior away from the Dirichlet prior 
corresponding to copy number two, 
the more likely the data is caused by a CNV, and, 
the higher is the informative/non-informative (I/NI) call.
\method{cn.mops} detects a CNV in the DNA of an individual as a segment 
with high I/NI calls.
I/NI call based CNV detection guarantees a low false discovery rate (FDR)
 because wrong detections are
less likely for high I/NI calls.
We assume that the genome is partitioned into segments in which
reads are counted but which need not be of constant length throughout the
genome. For each of such an segment we build a model.
We consider the read counts $x$ at a certain segment of the genome,
for which we construct a model across samples. The model incorporates
both read count variations due to technical or biological noise and
variations stemming from copy number variations.
For further information regarding the algorithm and its assessment 
see the %\cnmops\ 
\method{cn.mops} homepage at
\url{http://www.bioinf.jku.at/software/cnmops/cnmops.html}.



\section{Getting started and quick start}

To load the package, enter the following in your \R\ session:
<<echo=TRUE>>=
library(cn.mops)
@

The whole pipeline will only take a few steps, if BAM files are available 
(for read count matrices directly go to step 2):

\begin{enumerate}
\item Getting the input data from BAM files (also see Section \ref{s:bam} and
Section \ref{s:input}).
<<eval=FALSE>>=
BAMFiles <- list.files(pattern=".bam$")
bamDataRanges <- getReadCountsFromBAM(BAMFiles)
@

\item Running the algorithm (also see Section \ref{s:cn.mops}).
<<eval=FALSE>>=
res <- cn.mops(bamDataRanges)
@

\item Visualization of the detected CNV regions. For more information about 
the result objects and visualization see Section \ref{s:cn.mops}.
<<eval=FALSE>>=
plot(res,which=1)
@


<<fig=FALSE,echo=FALSE,results=hide>>=
data(cn.mops)
resCNMOPS <- cn.mops(XRanges)
pdf("003.pdf")
plot(resCNMOPS,which=7,toFile=TRUE)
dev.off()
@

\begin{figure}[H]
\begin{center}
\includegraphics[angle=0,width= 0.9\columnwidth]{003.pdf}
\end{center}
\end{figure} 
\end{enumerate}

\section{Input of cn.mops: BAM files, GRanges objects, or numeric matrices} %!!!!!
\label{s:input}
\subsection{Read count matrices as input}
\cnmops\ does not require the data samples to be of any
specific kind or structure. \cnmops\ only requires a {\em read count matrix}, 
i.e., given $N$ data samples and $m$ genomic segments, this is an $m\times N$ 
real- or integer-valued matrix $\mathbf{X}$,
in which an entry $x_{ij}$ corresponds to the read count of sample $j$ in the
$i$-th segment. E.g. in the following read count matrix sample three has
$17$ reads in the second segment: $x_{23}=71$.


\newlength{\mylen} 
\setlength{\mylen}{0.43cm}

\[\mathbf{X}= \begin{array}{c} \\ \mathrm{Segment\ 1} \\ \mathrm{Segment\ 2} \\ 
\mathrm{Segment\ 3} \\ \mathrm{Segment\ 4}\\ \mathrm{Segment\ 5} \\ 
\mathrm{Segment\ 6} \\ \hspace{0.2cm} \end{array}               
\begin{array}{c}
\begin{array}{cccc}
\mathrm{Sample\ 1} & \mathrm{Sample\ 2} & \mathrm{Sample\ 3} & 
\mathrm{Sample\ 4}\end{array}\\
\left(\begin{array}{cccc}
\hspace{\mylen}88\hspace{\mylen} & \hspace{\mylen}82\hspace{\mylen} & 
\hspace{\mylen}79\hspace{\mylen} & \hspace{\mylen}101\hspace{\mylen}\\
83 & 78 & 71 & 99\\
43 & 50 & 55 & 37\\
47 & 58 & 48 & 42 \\
73 & 86 & 95 & 91\\
92 & 90 & 80 & 71 
\end{array}\right) \\ \hspace{0.2cm}  \end{array}
\]


\cnmops\ can handle numeric and integer matrices or \verb+GRanges+ objects, 
where the read counts are stored as \verb+values+ of the object. 



\subsection{BAM files as input}
\label{s:bam}
The most widely used file format for aligned short reads is the Sequence 
Alignment Map (SAM) format or in the compressed form the Binary Alignment Map 
(BAM). We provide a simple function that makes use of the \texttt{Rsamtools} 
package to obtain the alignment positions of reads. The result object of the
function can directly be used as input for \cnmops. The author can provide 
functions for input formats other than BAM upon request: 
\email{cn.mops@bioinf.jku.at}.

<<>>=
BAMFiles <- list.files(system.file("extdata", package="cn.mops"),pattern=".bam$",
		full.names=TRUE)
bamDataRanges <- getReadCountsFromBAM(BAMFiles,
		sampleNames=paste("Sample",1:3),mode="unpaired")
@

In \verb+bamDataRanges+ you have now stored the genomic segments (left of the 
$\mid$'s) and the read counts (right of the $\mid$'s):
<<>>=
(bamDataRanges)
@


\section{Copy number estimation with cn.mops}
\label{s:cn.mops}
To get a first impression, we use a data set, where CNVs have been
artificially implanted. The simulated data set was generated using
distributions of read counts as they appear in real sequencing experiments. 
CNVs were implanted under the assumption that the expected read count is linear
dependent on the copy number. For example in a certain genomic we expect
$\lambda$ reads for copy number 2, then we expect $2\lambda$ reads for 
copy number 4.
The linear relationship was confirmed in different studies, like 
\citet{Alkan:09}, \citet{Chiang:09} and \citet{Quackenbush:11}.
 
\subsection{Running cn.mops}
 The read counts are stored in the objects \verb+X+ and
\verb+XRanges+, which are the two basic input types that \cnmops\ allows:
<<echo=TRUE>>=
data(cn.mops)
ls()
@

The same data is stored in a \verb+GRanges+ object, in which we see the
genomic coordinates, as well as the read counts (values):
<<>>=
head(XRanges[,1:3])
@
We are now ready to run \cnmops\ on the \verb+GRanges+ object:
<<eval=FALSE>>=
resCNMOPS <- cn.mops(XRanges)
@



Alternatively, it is possible to use an integer matrix, where the genomic
coordinates can be stored as \verb+rownames+ and the entries are the read counts. 
For example the data from above represented by an integer matrix \verb+X+:
<<>>=
head(X[,1:3])
@
We are now ready to run \cnmops\ on the integer matrix:
<<eval=FALSE>>=
resCNMOPSX <- cn.mops(X)
@


Note that the two results \verb+resCNMOPS+ and \verb+resCNMOPSRanges+ identify
the same CNVs: 
<<eval=FALSE>>=
all(individualCall(resCNMOPSX)==individualCall(resCNMOPS))
@

\subsection{The result object}
To get a summary of the CNV detection result, just enter the name of the
object (which implicitly calls \verb+show+):
<<eval=FALSE>>=
(resCNMOPS)
@

The CNVs per individual are stored in the slot \verb+cnvs+:
<<>>=
cnvs(resCNMOPS)[1:5]
@

Segments, in which individual CNVs accumulate, are called CNV regions and can be
accessed by \verb+cnvr+:
<<>>=
cnvr(resCNMOPS)[1,1:5]
@

We now want to check, whether \cnmops\ found the implanted CNVs. 
We have stored the implanted CNVs (see beginning of Section \label{s:cn.mops}) 
in the object \verb+CNVRanges+.
<<echo=TRUE>>=
(CNVRanges[15,1:5])
@

Next we identify overlaps between CNVs that were detected by \cnmops\ and
CNVs that were implanted. Towards this end we use the functions 
of the \texttt{GenomicRanges} package.
<<>>=
ranges(cnvr(resCNMOPS))[1:2]
ranges(cnvr(resCNMOPS)) %in% ranges(CNVRanges)
@ 

The function \verb+cn.mops+ creates an instance of the S4
class \verb+CNVDetectionResult+ that is defined by the present package. To get
detailed information on which data are stored in such objects, enter
<<eval=FALSE>>=
help(CNVDetectionResult)
@

\section{Visualization of the result}
\subsection{Chromosome plots} %!!!!
\cnmops\ allows for plotting the detected segments of an individual at one 
chromosome by a plot similar to the ones produced by \texttt{DNAcopy}:

\begin{center}
<<eval=FALSE>>=
segplot(resCNMOPS,sampleIdx=13)
@

<<fig=FALSE,echo=FALSE,results=hide>>=
pdf("002.pdf")
segplot(resCNMOPS,sampleIdx=13)
dev.off()
@

\begin{figure}[H]
\begin{center}
\includegraphics[angle=0,width= \columnwidth]{002}
\caption{The x-axis represents the genomic position and on the y-axis we 
see the log ratio of the read counts (green) and the copy number call
of each segment (red).}
\end{center}
\end{figure} 
\end{center}



\subsection{CNV region plots} %!!!!
\cnmops\ allows for plotting the detected CNV regions:

\begin{center}
<<eval=FALSE>>=
plot(resCNMOPS,which=1)
@

<<fig=FALSE,echo=FALSE,results=hide>>=
pdf("001.pdf")
plot(resCNMOPS,which=1,toFile=TRUE)
dev.off()
@

\begin{figure}[H]
\begin{center}
\includegraphics[angle=0,width= \columnwidth]{001}
\caption{The x-axis represents the genomic position and on the y-axis we 
see the read counts (left), the call of the local model (middle) and the
CNV call produced by the segmentation algorithm. Blue lines mark samples having
 a copy number loss.}
\end{center}
\end{figure} 
\end{center}

%In the left plot we see the read counts, where one line corresponds to the read
%counts of one sample along the genomic region displayed below in the subtitle.
%In the middle plot the local assessments (signed individual I/NI calls)
%before applying the segmentation algorithm are displayed and on the right the
%final CNV call is shown. 




\section{Adjusting sensitivity, specificity and resolution for specific
applications}
The default parameters of both the local models of \cnmops and the segmentation
algorithm were optimized on a wide ranged of different data sets. However, you 
might want to adjust sensitivity and specificity or resolution to your specific
needs.

\begin{itemize}
\item[\tt upperThreshold] The calling threshold for copy number 
gains, a positive value.  
Lowering the threshold will increase the detections, raising will decrease
 the detections.
\item[\tt lowerThreshold] The calling threshold for copy number 
losses, a negative value. Raising the threshold will increase the detections, 
lowering will decrease the detections.
\item[\tt priorImpact] This parameter should be optimized for each data set, 
since it is influenced by number of samples as well as noise level. 
The higher the value, the more samples will have copy number 2, and 
consequently less CNVs will be detected. 
\item[\tt minWidth] The minimum length of CNVs measured in number of segments.
The more adjacent segments with a high or low copy number call are joined, the
higher the confidence in the detections.  A lower  value will lead to more
shorter segments, and a higher value will yield to less, but longer segments.
\end{itemize}

The length of the initial segments is also crucial. They should be 
chosen such that on average 100 reads lie in one segment. The {\tt WL} 
parameter of {\tt getReadCountsFromBAM} determines this resolution.


\section{Exome sequencing data}
To apply \cnmops\ to exome sequencing data requires a different preprocessing,
since constant windows spanning the whole genome are not appropiate. The initial
segments in which the reads are counted should be chosen as the regions of
the baits, targets or exons. The read count matrix can now be generated by 
using the function {\tt getSegmentReadCountsFromBAM} that requires the 
genomic coordinates of the predefined segments as {\tt GRanges} object.
The resulting read count matrix can directly be used as input for \cnmops.
A possible processing script could look like the following:

<<eval=FALSE>>=
library(cn.mops)
BAMFiles <- list.files(pattern=".bam$")
segments <- read.table("targetRegions.bed",sep="\t",as.is=TRUE)
gr <- GRanges(segments[,1],IRanges(segments[,2],segments[,3]))
X <- getSegmentReadCountsFromBAM(BAMFiles,GR=gr,mode="unpaired")
resCNMOPS <- cn.mops(X)
@

\paragraph{Possible issues and notes}
A problem can occur, if the names of the reference sequences, e.s. chromosomes,
are inconsistent between the bed file and the bam file. For example "chr1", 
"chr2",...,"chrX","chrY" and "1","2",...,"X","Y".This can easily be solved by
replacing the seqlevels of the GRanges object:
<<eval=FALSE>>=
#the following removes the "chr" from reference sequence names
seqlevels(gr) <- gsub("chr","",seqlevels(gr))
@

Results can also be improved if you extend your target regions by a small 
amount of bases to the left and to the right (in the following case it is
30bp):
<<eval=FALSE>>=
gr <- GRanges(segments[,1],IRanges(segments[,2]-30,segments[,3]+30))
gr <- reduce(gr)
@


\section{Heterosomes and CNVs of tumor samples}
With the default settings the normalization procedure assumes that the ploidy
of each sample is the same. However, it is possible to account for different karyotypes. 
When analyzing CNVs on the X or Y chromosome one possibility is to treat males and 
females separately. The second option is to provide the normalization function 
with the information about the gender, that is different ploidy states of the X and Y chromosome. 
This can be handled by the {\tt ploidy} parameter of the normalization function.
In the following we show the normalization for the X chromosome, 
if the first 10 individuals are males ({\tt ploidy} set to 1) and the next
30 individuals are females ({\tt ploidy} set to 2):

<<>>=
XchrX <- normalizeChromosomes(X[1:500, ],ploidy=c(rep(1,10),rep(2,30)))
cnvr(cn.mops(XchrX,norm=FALSE))
@

Karyotype information can also improve results of CNV detection in tumor 
samples. The best results can be reached, if for each chromosome the number of
appearances in the cell is known. In this case normalization should be applied 
to each chromosome separately. 


\section{cn.MOPS for haploid genomes}
For haploid genomes the prior assumption is that all samples have copy number 1.
The function {\tt haplocn.mops} implements the cn.MOPS algorithm adjusted to
haploid genomes. 

<<eval=FALSE>>=
resHaplo <- haplocn.mops(X)
@





\section{How to cite this package}

If you use this package for research that is published later, you are kindly
asked to cite it as follows:
\citep{Klambauer:11}.

To obtain Bib\TeX\ entries of the reference, you can enter the following
into your R session:
<<eval=FALSE>>=
toBibtex(citation("cn.mops"))
@ 

\bibliographystyle{natbib}
\bibliography{cnv}


\end{document}
