%\VignetteIndexEntry{An RRBS data set with 12 samples and 10,000 simulated DMRs.}
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\begin{document}
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\title{RRBSdata: An RRBS data set with simulated DMRs.}
\author{Katja Hebestreit, Hans-Ulrich Klein}
\date{October 10, 2014}

\maketitle

\tableofcontents

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\section{Introduction}
This package contains RRBS data with simulated differentially
methylated regions (DMR's). In particular, it comprises 12 samples that are
divided into 'cancer' and 'control' samples. We simulated 10,000 DMRs
with different lengths and differences. This data set was used for
\cite{Klein2014} and can be used for any method evaluation to find DMRs.

\section{Simulation}
Instead of simulating bisulfite sequencing data, we used a real data
set in which we incorporated DMRs. This procedure has the advantage
that technical and biological characteristics of bisulfite sequencing
data are present in the simulation data, for example, the correlation
of DNA methylation of nearby CpG sites. And, especially, biological
and technical DNA methylation variation across CpG sites and across samples is
preserved. To obtain a dataset with known DMRs, we used 12 human control samples
from a previously published RRBS data set
\cite{Schoofs2012}. We simulated DMRs of different lengths and
intensities by altering the number of methylated reads of some of the
CpG sites in half of the samples, which could be considered as the cancer
samples. We downloaded CpG island positions from UCSC database and
filtered out all islands that were not covered in any of the samples
(27\,718 remaining islands). Only islands with not less than 10
covered CpG sites were considered to receive a DMR (24,698
islands). Within these 24,698 CpG
islands we incorporated 10\,000 DMRs with methylation differences of
10, 20, 30, or 40\%. The DMRs spanned 10, 20, 30, or
40\% of the CpG sites of the respective islands. We made sure that
we gained the same amount of DMRs for each of
the 16 combinations of methylation difference and percentage of
modified CpG sites, that is, we gained 1\,250 DMRs per combination.

We incorporated the DMRs as follows:
1.) We devided the 12 control samples into 6 ``cancer'' samples
and 6 ``control'' samples.
2.) Within each CpG island we only considered regions to receive a
DMR if each of its CpG sites was covered in at least half of all
samples. Those regions are referred to as ``covered island regions''.
3.) For each CpG site within covered island regions we determined its
minimum and maximum smoothed methylation level across all samples.
4.) For each CpG island we determined the maximum percentage of
neighbored CpG sites that can be altered by determining the percentage of CpG sites within
its biggest covered island region on all CpG sites
within the island.
5.) Each of the 10,000 DMRs was sampled into a covered island region
of a CpG island that was appropriate to harbor the DMR, in terms of minimum and maximum
DNA methylation and CpG percentage of its largest covered island
region on all CpG sites within the island. Not more than one DMR was incorporated per CpG island.
6.) Whenever it was possible to increase the DNA methylation by the amount of
the difference of the respective DMR (that is, the resulting
methylation level is below 1), it was increased in the cancer
samples. Otherwise, the DNA methylation was decreased by the amount of
difference.
7.) To increase the DNA methylation in a cancer sample within a region by a certain
amount, the number of methylated reads of the respective CpG sites was
increased. For instance, a CpG site of coverage 12 with 3 methylated
reads (and a relative methylation level of 0.25) within a region
that should get a methylation difference of 0.3, received 4 additional
methylated reads (because: 0.3x12 = 4).

\section{Data objects}
There are three data objects: \Robject{rrbs}, \Robject{islands} and \Robject{diffMethCpGs}.

The \Robject{rrbs} object is a \Rclass{BSraw-class} object from the
\Rpackage{BiSeq} package. It comprises the RRBS data:

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library(BiSeq)
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<<>>=
data(rrbs)
rrbs
head(colData(rrbs))
@

The \Robject{islands} object is a \Rclass{GRanges-class} object comprising
all CpG islands that were considered to contain DMRs. Please see
\Rcode{?islands} for information regarding the columns.

The \Robject{diff.meth.cpgs} object is a \Rclass{GRanges-class} object
comprising all differentially methylated CpG sites:

<<>>=
data(diffMethCpGs)
@


\bibliographystyle{unsrturl}
\bibliography{RRBSdata}


\end{document}