%\VignetteIndexEntry{RNA-Seq Tutorial}
%\VignetteKeywords{RNA-Seq}
%\VignettePackage{RnaSeqTutorial}
\documentclass[11pt]{article}
\usepackage{verbatim}
\usepackage{natbib}
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\title{RNA-Seq Tutorial (EBI, October 2011)}
\author{Nicolas Delhomme}

\SweaveOpts{keep.source=TRUE}

\begin{document}

\maketitle

\tableofcontents

\begin{comment}
Just a comment example.
TODO turn the eval back to tru on line 534 and 918 once rtracklayer is
working again for R-2.15
\end{comment}

\newpage

\section{Introduction}

This file describes a RNA-Seq analysis
use-case. RNA-Seq \citep{Mortazavi:2008p740} was introduced as a new
method to perform Gene Expression Analysis, using the advantages of
the high throughput of \emph{Next-Generation Sequencing} (NGS)
machines. The goal of this use-case is to generate a count table for
the selected genic features of interest, \emph{i.e.} exons, transcripts, gene
models, \emph{etc.}
\newline

In the first part, the data will be read in R using Bioconductor
\citep{Gentleman:2004p2013} \Rpackage{ShortRead}
\citep{Morgan:2009p739}, \Rpackage{Rsamtools} and
\Rpackage{GenomicRanges} packages. Then, annotation will be retrieved
using the \Rpackage{biomaRt} \citep{Durinck:2005p1990} and
\Rpackage{genomeIntervals} packages. Finally the \Rpackage{IRanges}
and \Rpackage{GenomicFeatures} packages will be used to define the
reads coverage, and assign counts to genic features of interest.
\newline

In the second part, we will see how this process can be simplified by
the use of the \Rpackage{easyRNASeq} package \citep{Delhomme:2012p4678} and how more advanced
pre-processing can be performed, such as \emph{de-multiplexing},
\emph{RPKM} ``correction'' or normalization using the \Rpackage{DESeq}
or \Rpackage{edgeR} packages. 
\newline

Finally, the count information will be exported as bed and wig
formatted file, to be visualized into the UCSC genome browser or a
stand alone genome browser like IGB.
\newline

The overall process is described in figure \ref{fig:fig1}, page
\pageref{fig:fig1}. First, the genomic and genic annotation will be
retrieved from the selected/preferred source and converted into an
appropriate \Robject{object}. In parallel, the sequenced reads'
information (\textit{e.g.} chromosome, position, strand,
\textit{etc.}) will be retrieved from the alignment file and, as well,
converted to a similar \Robject{object}. Then, the reads contained in
the \Robject{reads} object are summarized per genic annotation
contained in the \Robject{annotation} object. This give raise to a
count table that, finally, can be normalized using additional R
packages. 

\begin{figure}[htpb]
\centerline{\includegraphics[width=0.80\textwidth]{RnaSeqProcess.png}}
\caption{RNA-Seq Procedure Overview. The R packages used for the
  different steps are emphasized in bold face.}
\label{fig:fig1}
\end{figure}

\newpage

\section{Walk through a single sample use case}

In this section, we will mainly look into the details of generating
a count table, \emph{i.e.} how many sequencing reads can be assigned to
given genic features. An expressed genic feature can be anything from
an exon to a gene-model or, as recently published,
enhancers \citep{Kim:2010p572}. In this section, you will learn how to
read ``raw'' data in, load the related annotations, calculate the
reads genomic coverage and deduce read counts per exons. But first, we
need to load the tutorial library and data.

<<Lood the tutorial library>>=
library(RnaSeqTutorial)
@ 

\newpage

\subsection{Reading the data}

There are different ways to read in NGS data into R, depending on the
``raw'' data format at hand. The next paragraphs will present three
different approaches, introducing the \Rpackage{ShortRead},
\Rpackage{Rsamtools} and \Rpackage{GenomicRanges} packages capabilities.

\paragraph{ShortRead}

\Rpackage{ShortRead} was the first NGS package developed to read in
NGS data and is able to read almost every sequencer's manufacturer
proprietary formats (with the notable exception of ABI
color-space). First, an Illumina ``export'' file produced by a GenomeAnalyzer
GAIIx will be read in; and then the same data set, in BAM format.
\subparagraph{Illumina export}
The export file is read in using the \Rfunction{readAligned} function,
and the resulting object displayed. 

<<Read the data, eval=TRUE>>=
library(ShortRead)
aln<-readAligned(
                 system.file("extdata",package="RnaSeqTutorial"),
                 pattern="subset_export",type="SolexaExport")
show(aln)
@ 

\emph{N.B.}: The \Rfunction{system.file} retrieves the file path where
a package was installed. Additional argument can be provided in a
similar fashion to that of \Rfunction{file.path} to access directories
and files within the package.

The \Rfunction{readAligned} function returns an object of the
\Rclass{AlignedRead} class. The main slots can be accessed using
similarly named accessors as described in the following code sample:

<<The AlignedRead object, eval=FALSE>>=
chromosome(aln)
levels(chromosome(aln))
position(aln)[1:100]
width(aln)[1:100]
strand(aln)[1:100]
sread(aln)
quality(aln)
@ 

The Illumina ``export'' format contains every read sequenced on the
platform, as well as these coming from overlapping sequence
clusters. These sequences are flagged by the Illumina pipeline through
a \emph{chastity filter}. It is important for processing such ``raw''
data to remove these reads. This will be done in the next section:
\ref{sec:filt}, page \pageref{sec:filt}. First, we will just find out the value of the chastity
filter field (``Y'' or ``N'' whether it passes the filter or not)
within the current object: 

<<The chastity field, eval=FALSE>>=
alignData(aln)$filtering[1:100]
@

\subparagraph{BAM}

The SAM/BAM format \citep{Li:2009p1662} is becoming a \emph{de-facto}
standard for storing NGS aligned data. As a consequence, the
\Rpackage{Rsamtools} was developed to import the \emph{samtools}
functionalities into the R environment. Subsequently, the
\Rpackage{ShortRead} package was extended to use \Rpackage{Rsamtools}
to load BAM files. SAM/BAM files can be sorted by chromosomal
position, in which case an index can be created that will improve the
subsequent retrieval of information within the BAM file. This can be
done using the ``samtools'' command in a terminal, or using the
Rsamtools package that implements in R most of the ``samtools'' and
``bcftools'' functionalities. In the following example, you will first
create an index for the BAM file and then read the data into an
\Rclass{AlignedRead} class object. 

<<Indexing a BAM file with ShortRead, eval=TRUE>>=
library(Rsamtools)
file.copy(
          system.file("extdata",
                      "subset.bam",
                      package="RnaSeqTutorial"),
          getwd())
indexFile <- indexBam("subset.bam")
basename(indexFile)
@

<<Reading a bam file>>=
aln2 <- readAligned(getwd(),pattern="subset.bam$",type="BAM")
@ 

\textbf{Q1}: What differences exists between the \Robject{aln} and \Robject{aln2} objects?
\label{question1}
\newline
\newline
\textbf{Answers} are provided in the Appendix \ref{apdxA}, page \pageref{apdxA}.


\paragraph{Rsamtools}
A different, more flexible way to load BAM formatted files is to
directly use the \Rfunction{scanBam} function from the
\Rpackage{Rsamtools} package. 

<<Loading a file using Rsamtools>>=
aln2b <- scanBam(
                 "subset.bam",
                 index="subset.bam"
                 )
names(aln2b[[1]])
@ 

\emph{N.B.} First, the \emph{index} argument is the filename, \emph{i.e.} the ``.bai''
extension is ignored. Second, \Rfunction(scanBam) returns a list of
list, with the first list having a single element.

The inner lists contains an element per column of the BAM file
formats, with the optional fields being ignored. The
\Rfunction{scanBam} function extends the base R ``scan''
function. Additional parameters can be provided to select the reads:
see the \Robject{scanBamParam} argument for filtering the reads based
on their flag, cigar string, \emph{etc.}; see the
\Rfunction{ScanBamParam} to display which fields of the BAM file are
to be retrieved.

\paragraph{GenomicRanges}
Finally, the last example to load data uses the
\Rpackage{GenomicRanges} package. With the increasing read length, it
became possible to reliably map exon-exon junctions and therefore to
report \emph{gapped alignments} and alternative transcripts. Tools such
as TopHat \citep{Trapnell:2009p156} have especially been developed for that
purpose. The \Rpackage{GenomicRanges} package was implemented to deal with
this new kind of data. As of today, most of the common aligners such as
bowtie \citep{Langmead:2009p309}, bwa \citep{Li:2009p1660}, novoalign
\citep{ref:Novoalign} or GSNAP \citep{Wu:2010p2657} supports gap
alignments, an additional motivation to use the
\Rpackage{GenomicRanges} package functionalities.

<<Reading a Tophat BAM file>>=
library(GenomicRanges)
aln3 <- readGAlignments(
                        system.file("extdata",
                                    "gapped.bam",
                                    package="RnaSeqTutorial"),
                        format="BAM")
@ 

The generated object of class \Rclass{GAlignments}, is very
different from the \Rclass{AlignedRead} class objects you have seen so
far.
\newline
\newline
\textbf{Q2} What are the most obvious differences?
\label{question2}
\newline
\textbf{Q3}: Does the \Robject{aln3} object contains evidence of exon-exon junctions?
\newline
Tip: use the cigar functionalities: \Rfunction{cigar},
\Rfunction{cigarOpTable}, \emph{etc.}
\label{question3}

\paragraph{Caveats}

Finally, before we go on with data filtering, a few caveats need to be
mentioned.

\subparagraph{Illumina export}
The \Rpackage{ShortRead} package, when loading an export file does not
retrieve all the possible information; \emph{i.e.} the \Rfunction{id} slot of
the \Rclass{AlignedRead} object contains no valid information.

<<missing ids>>=
head(id(aln))
@ 

The \emph{withMultiplexIndex}, \emph{withPairedReadNumber},
\emph{withId} and \emph{withAll} arguments offer the possibility to retrieve
such information.
<<with ids>>=
aln4<-readAligned(
                  system.file("extdata",package="RnaSeqTutorial"),
                  pattern="subset_export",type="SolexaExport",
                  withId=TRUE)
head(id(aln4))
@ 

\subparagraph{BAM}
In a BAM file, the reads are stored as they are aligned against the
reference genome! Hence, these are not necessarily the ``sequence''
that have been read by the sequencer. The \Rpackage{ShortRead}, when
loading a BAM file revert the reads to their original sequence.

<<BAM read conversion>>=
sel <- !is.na(strand(aln2)) & strand(aln2) %in% "-"
aln2b[[1]]$seq[sel][1]
sread(aln2[sel])[1]
reverseComplement(aln2b[[1]]$seq[sel][1])
@ 

\paragraph{Conclusion}

In that section, we have seen three packages that allow loading NGS
data into R, as well as some caveats related to the format these data
can be in. Depending on the data format, this step might only be the
first one and some additional pre-processing might be necessary, as
described in the next paragraph.

\newpage

\subsection{Filtering the data}
\label{sec:filt}
As one can see, many reads do not pass the chastity filter and many
reads do not align to the genome. In addition some of those reads do
contain many \textbf{N}s; that is whenever Bustard, the Illumina base caller,
could not perform a valid base call. All the chastity flagged reads
should be filtered out. The N-containing reads can be filtered out
according to the number of mismatch you are willing to have in your data.
Filtering for failed chastity calls is not implemented in the
\Rpackage{ShortRead} package, but is in the \Rpackage{easyRNASeq}
package. In the following example, several filters are combined to
keep reads that align to reference chromosomes, that do not have more
than 2 Ns and that pass the chastity filter.

<<Filtering the reads, eval=FALSE>>=
library(easyRNASeq)
nFilt <- nFilter(2)
chrFilt <- chromosomeFilter(regex="chr")
cFilt <- chastityFilter()
filt <- compose(nFilt,chrFilt,cFilt)
aln <- aln[filt(aln)]
@ 

\begin{comment}
  We do not depend on easyRNASeq, so we cannot expect to be able to
  load the library. easyRNASeq depends on RnaSeqTutorial and we do not 
  want cyclic dependencies.
\end{comment}

<<Really filter but do not show, eval=TRUE, echo=FALSE>>=
nFilt <- nFilter(2)
chrFilt <- chromosomeFilter(regex="chr")
filt <- compose(nFilt,chrFilt)
aln <- aln[filt(aln)]
aln <- aln[alignData(aln)$filtering=="Y"]
@ 

<<the filtered aln, eval=FALSE>>=
show(aln)
@ 

We are now left with 56,883 ``valid'' reads, which we want to assign
to their respective exon. For this we need to get the proper genomic
and genic information. 

<<cleanup, eval=TRUE, echo=FALSE>>=
rm(aln2,aln2b,aln3,aln4)
silent<-gc(verbose=FALSE)
@ 

\paragraph{Conclusion}

We've seen how to load in R the raw data (the aligned reads). We
therefore have the information where these reads are located in the
genome. Now, we want to discover if these loci covered by reads
corresponds to interesting genomic or genic features, \emph{e.g.}
exons, promoters, \emph{etc.}. To achieve this, we first need to load
the genic/genomic annotation in R. This will be the topic of the next
subsection. 

\newpage

\subsection{Loading the annotation}

To assign reads to exons, we need to know the genome composition,
\emph{i.e.} how many chromosomes, their names and sizes. In addition, we need
to know the genic information, \emph{i.e.} where are exons located, which
gene they belong to, \emph{etc.}

\paragraph{Genomic information}
The reads present in the ``subset'' file used previously, come from an
RNA-Seq experiment conducted in \emph{Drosophila melanogaster}. First,
the genomic information for that organism need to be retrieved. You
will use the \Rpackage{BSgenome} package for this.

<<Getting the chromosome names and sizes, eval=TRUE>>=
library(BSgenome.Dmelanogaster.UCSC.dm3)
chrSizes <-seqlengths(Dmelanogaster)
chrSizes
@ 

\paragraph{Genic information}
To retrieve the genic annotation, several solutions are available:
\begin{itemize}
  \item the \Rpackage{biomaRt} package to fetch information from
    ``Mart'' databases
  \item the \Rpackage{genomeInterval} package to read data in
    from ``gff'' annotation files obtained from FlyBase
    \citep{Tweedie:2009p2014}, Ensembl \citep{Flicek:2011p2042},
    UCSC, or using proprietary annotations.
  \item the \Rpackage{rtracklayer} package to import ``gff'', ``bed'' or
    ``wig'' files.
  \item the \Rpackage{GenomicFeatures} package to retrieve information
    from the UCSC databases or from the ``Mart'' databases through the
    \Rpackage{biomaRt} package interface.
\end{itemize}

These four methods have strengths and drawbacks and selecting the
most appropriate is essentially depending on your computing
environment, \emph{e.g.} lots of memory vs. lots of disk space as well as on
your computing proficiency. Shortly, the two first approaches will require the
post-processing of the obtained data into a \Rclass{RangedData} or
\Rclass{GRanges} class object. The first and last require an internet
connection, at least for initially downloading the data. Such data can
be saved locally, not to have to download them again and again. Having
a local frozen copy is anyway a good practice to ensure
reproducibility. The last one requires to write an \emph{SQLite}
database locally. The two last returns object that do not need
post-processing; however the \Rpackage{rtracklayer} \Rfunction{import}
function is not robust to incorrect gff files and the
\Rpackage{GenomicFeatures} is limited to the genomes available in its
datasources.


\subparagraph{biomaRt}
The \Rpackage{biomaRt} package remotely queries Mart services. To get
the \emph{Drosophila melanogaster} genomic information, a connection
is established with the Ensembl fruit fly Mart database and queried
for the information we need (gene ID, transcript ID, exon ID, position,
\emph{etc.}). To limit the amount of data to be retrieved, a filter is
set to select for the chromosomes of interest. The
\emph{filters} argument defines the criteria to use as filters and
the \emph{values'} one defines the values that are accepted for these
criteria.

<<Exon Transcript Annotation>>=
library(biomaRt)
ensembl <- useMart("ensembl",
                   dataset="dmelanogaster_gene_ensembl")
exon.annotation<-getBM(
                       c("ensembl_gene_id",
                         "strand",
                         "ensembl_transcript_id",
                         "chromosome_name",
                         "ensembl_exon_id",
                         "exon_chrom_start",
                         "exon_chrom_end"),
                       mart=ensembl,
                       filters="chromosome_name",
                       values=c("2L","2R","3L","3R","4","X"))
@ 

As mentioned, the obtained \Robject{data.frame} needs to be converted
into an object of class \Rclass{RangedData} or \Rclass{GRanges}. Note
that the chromosome names retrieved from Ensembl, compliant with the
\emph{FlyBase} annotation are not UCSC compliant and need to be
converted; \emph{i.e.} the ``chr'' string needs to be prepended.

<<Exon Transcript object transformation>>=
exon.annotation$chromosome <- paste(
                                    "chr",
                                    exon.annotation$chromosome_name,
                                    sep="")
exon.range <- RangedData(
                         IRanges(
                                 start=exon.annotation$exon_chrom_start,
                                 end=exon.annotation$exon_chrom_end),
                         space=exon.annotation$chromosome,
                         strand=exon.annotation$strand,
                         transcript=exon.annotation$ensembl_transcript_id,
                         gene=exon.annotation$ensembl_gene_id,
                         exon=exon.annotation$ensembl_exon_id,
                         universe = "Dm3"
                         )
@

This created a \Rclass{RangedData} class object.

<<Check the objects, eval=FALSE>>= 
show(exon.range)
@ 

\textbf{Q4}: How would you create a \Rclass{GRanges} object?
\label{question4}

\subparagraph{genomeIntervals}

The \Rfunction{readGff3} of the \Rpackage{genomeIntervals} is a robust
and efficient way to load Generic Feature Format (gff) file. The
latest version of that format: \emph{version 3} is used by most model
organism websites, or similar format have been derived from it, such
as the Gene Transfer Format (gtf) used among others by Ensembl. The
\Rfunction{readGff3} is flexible enough to cope with gtf formatted
files too.

<<Read in the gff>>=
library(genomeIntervals)
gInterval<-readGff3(system.file("extdata",
                                "annot.gff",
                                package="RnaSeqTutorial"))
@ 

As for \Rpackage{biomaRt}, post-processing the obtained object is
necessary. The gff file has been retrieved from \emph{FlyBase} and
filtered for the ``exon'' type. It contains the \textit{gffAttributes}
\textit{ID}, \textit{Name} and \textit{Parent} defining the
``exon ID'', the ``gene ID'' and the ``transcript ID'' respectively.

<<post process the gff>>=
exon.range2 <- RangedData(
                          IRanges(
                                  start=gInterval[,1],
                                  end=gInterval[,2]),
                          space=gInterval$seq_name,
                          strand=gInterval$strand,
                          transcript=as.vector(
                            getGffAttribute(gInterval,"Parent")),
                          gene=as.vector(
                            getGffAttribute(gInterval,"Name")),
                          exon=as.vector(
                            getGffAttribute(gInterval,"ID")),
                          universe = "Dm3"
                          )
@ 

\textbf{Q5}: How would you create a \Rclass{GRanges} object from the \Robject{gInterval}?
\label{question5}

\textbf{Q6}: (optional) How would you \emph{export} the ``gAnnot.rda'' file,
present in the ``data'' directory into a gff version \emph{3} formatted file?
\label{question6}

\subparagraph{rtracklayer}

Importing a properly formatted ``gff'' file is straightforward.

<<importing with rtracklayer, eval=FALSE>>=
library(rtracklayer)
exon.range3<-import.gff3(
                    system.file("extdata",
                                "annot.gff",
                                package="RnaSeqTutorial")
                    )
@ 

\subparagraph{GenomicFeatures}

The \Rpackage{GenomicFeatures} can retrieve data by connecting
``UCSC'' or by through the \Rpackage{biomaRt} package interface. The
two related functions are \Rfunction{makeTranscriptDbFromUCSC} and
\Rfunction{makeTranscriptDbFromBiomart}. It creates a TranscriptDb
object that can be queried using the \Rfunction{transcript},
\Rfunction{exon} and \Rfunction{cds} functions.

<<importing with GenomicFeatures, eval=FALSE>>=
library(GenomicFeatures)
@ 

<<GenomicFeature taste, eval=FALSE>>=
dm3.tx <- makeTranscriptDbFromUCSC(
                                   genome="dm3",
                                   tablename="refGene")
exon.range4 <- exons(dm3.tx)
exon.range4
@ 

As you can see a certain amount of warnings are raised and the actual
annotation looks different (somewhat less complete) than the previous
ones. To avoid downloading the annotation everytime they  are needed,
the annotation object (\Robject{dm3.tx} in that case) can be saved to
disk. This is actually a good practice as well to ensure the
reproducibility of your analyses.

\subparagraph{Caveats}
All the previous steps help retrieve the necessary genomic and genic
information, however it is \textbf{essential} for the user to pay attention that
the proper annotation are gathered.
The \Rpackage{GenomicFeatures} is under very active development, so
changes are to be expected. If your genome is not in it,do not
hesitate to post on the mailing list,to make the changes happen!
It is as well \textbf{essential} that you understand the content of
your annotation and its consequences on your analysis.\textit{I.e.}
most of the previously described approaches will result in annotation
containing some overlapping genic feature, \textit{e.g.} genes on
opposite strand, exons shared by transcripts, \textit{etc.} Using them
as is will results in counting some reads multiple times, introducing
some possible bias. It is obviously important to avoid such behavior
and this requires to check and validate you annotation.

<<clean, echo=FALSE, eval=TRUE>>=
rm(exon.range,exon.range3,gInterval,ensembl,exon.annotation,sel)
silent<-gc(verbose=FALSE)
@ 

\newpage

\subsection{Summarizing read counts per feature}

Now that all the annotations have been retrieved (\Robject{exon.range2}
and \Robject{chrSizes}) and the data loaded (\Robject{aln}), the read
coverage can be extracted and then summarized per feature of
interest.

\paragraph{Calculating the coverage}

<<Genomic coverage, eval=TRUE>>=
cover <- coverage(aln,width=chrSizes)
@ 

<<Have a look at the coverage, eval=FALSE>>=
show(cover)
show(cover$chr2R)
@

\Rclass{RleList} objects are clever way of encoding a coverage
vector. Such a vector, contains one value per bp and is therefore
greedy. However many successive bp might have the same coverage value
and therefore could be considered as intervals that have a given
coverage value. This is exactly what an \Rclass{Rle} object does. It
is constituted of ``runs'' that encode the length of an interval
together with its coverage value.
\newline
\newline
\textbf{Q7}: How would you get the actual bp coverage from an \Rclass{Rle} class object?
\label{question7}
Tip: select the ``chr4'' out of your \Robject{cover} \Rclass{RleList};
it is the smallest.
\newline
\textbf{Q8}: How would you access the ``run'' lengths and values?
\label{question8}
\newline
\newline
The \Rclass{Rle} class derives from the \Rclass{Sequence} one and
therefore has numerous capabilities. The following example display
some: 

<<Additional commands for cov, eval=TRUE>>=
runLength(cover$chr4)[1:3]
runValue(cover$chr4)[1:3]
r.start <- runLength(cover$chr4)[1]+1
r.end <- sum(runLength(cover$chr4)[1:2])
as.integer(cover$chr4)[r.start:r.end]
as.integer(window(cover$chr4,r.start,r.end))
@

The previous example identifies a region covered by a single read and
retrieve the actual bp coverage from it. Note the use of
the \Rfunction{window} function. It significantly fastens such
approaches; \emph{i.e.} it works similarly to a
\Rclass{Views}. \Rclass{Views} is a general container for storing a
set of ``views'' on an object; \textit{i.e.} a view simply records the
position of interest on the object rather than creating a copy of that
object. For example, a view on a genomic sequence such as a chromosome
would simply register the start and the width rather than the actual sequence.

\paragraph{Aggregating the coverage per exon}

Now to convert the genomic coverage into an exonic one, we need to use
the genic annotation retrieved previously. The coverage object:
\Robject{cover} we have generated earlier  describes the number of
reads overlapping every bp in the genome. To summarize its values per
exon, the simplest approach is to average the coverage for all bp covered
by a given exon. 

<<Exon Transcript coverage, eval=TRUE>>=
exon.coverage<-aggregate(
                         cover[match(names(exon.range2),names(cover))],
                         ranges(exon.range2),
                         sum)

exon.coverage <- ceiling(exon.coverage/unique(width(aln)))
@ 

<<Ex. tr. cov.p.2, eval=FALSE>>=
show(exon.coverage)
@ 

\subparagraph{Tip}
Using Views actually make this approach much faster:
<<faster, eval=FALSE>>=
viewSums(
         Views(
               cover[match(names(exon.range2),names(cover))],
               ranges(exon.range2)))
@ 

\subparagraph{Caveats}

Note the \Rfunction{match} that is done between the
\Robject{names(exon.range2)} and \Robject{names(cover)}. 
\newline
\newline
\textbf{Q9}: Why is it so \textbf{essential}?
\label{question9}
\newline
\textbf{Q10}: List the possible drawback of this approach.
\label{question10}
\newline
\textbf{Q11}: Use the \Rfunction{countOverlaps} to overcome some of these limitations.
\label{question11}

\newpage

\subsection{Conclusion}

Now, you are done with processing that single sample. If you were to
do this for many samples, it would be demanding and probably not
ideally fail-safe. Rationalizing and automating that task was our
motivation to build the \Rpackage{easyRNASeq} package. However, keep
in mind that getting the proper annotation for your analysis is an
essential step, as well when using the \Rpackage{easyRNASeq}
package \citep{Delhomme:2012p4678}. You'll probably want to avoid
counting the same read multiple times, \textit{e.g.} in overlapping
exons. 

<<clean up, echo=FALSE>>=
rm(aln,chrSizes)
silent<-gc(verbose=FALSE)
@ 

\newpage

\section{Using easyRNASeq}

Let us redo what was done in the previous sections. Note that most of
the \Rclass{RNAseq} object slots are optional. However, it is advised
to set them, especially the \emph{readLength} and the
\emph{organismName}; to help having a proper documentation of your
analysis. The organismName slot is actually mandatory if you want to
get genomic annotation using \Rpackage{biomaRt}. In that case, you
need to provide the name as specified in the corresponding
\Rpackage{BSgenome} package, \emph{i.e.} ``Dmelanogaster'' for the
\Rpackage{BSgenome.Dmelanogaster.UCSC.dm3} package.

\subsection{easyRNASeq}

<<Load the library and create the object,eval=FALSE>>=
## load the library
library("easyRNASeq")
library(BSgenome.Dmelanogaster.UCSC.dm3)

count.table <- easyRNASeq(system.file(
                                      "extdata",
                                      package="RnaSeqTutorial"),
                          organism="Dmelanogaster",
                          chr.sizes=as.list(seqlengths(Dmelanogaster)),
                          readLength=36L,
                          annotationMethod="rda",
                          annotationFile=system.file(
                            "data",
                            "gAnnot.rda",
                            package="RnaSeqTutorial"),
                          format="bam",
                          count="exons",
                          pattern="[A,C,T,G]{6}\\.bam$")
@ 

<<Show, eval=FALSE>>=
head(count.table)
dim(count.table)
@ 

That is all. In one command, you got the count table for your 4 samples!

\paragraph{Warnings}
\label{p:W}
As you could see when running the previous example,  warnings were
emitted and quite rightly so. 
\begin{enumerate}
\item about the annotation:
The annotation we are using here is
redundant and this at two levels. First, some exons overlap. These are
alternative exons from different transcript isoforms. Second, the
annotation contains the information about all the possible different
transcript isoforms. This means that some exons are
duplicated. Therefore counting by exons or transcripts using these
annotation will result in counting some of the reads several
times. There might be reasons one might want to do that, but as it is
probably not what you want when performing an RNA-Seq analysis, the
warning is emitted. As this can be a very significant source of error,
all the examples here will emit this warning. The ideal
solution is to provide an annotation object that contains no
overlapping features. The \Rfunction{disjoin} function from the
\Rpackage{IRanges} package offers a way to achieve this.
\item about potential naming issue in the input file:
It is (sadly) very frequent that the sequencing facilities use
different naming conventions for the chromosomes they report in the
alignment files. It is therefore very frequent that the annotation
provided to easyRNASeq uses different chromosome names than the
alignment file. These warnings are there to inform you about this
issue.
\end{enumerate}

\paragraph{Details}
The \Rfunction{easyRNASeq} function currently accepts the following
\Robject{annotationMethods}: 
\begin{itemize}
  \item ``biomaRt'' use biomaRt to retrieve the annotation
  \item ``env'' use a \Rclass{RangedData} class object present in the environment
  \item ``gff'' reads in a gff version 3 file
  \item ``gtf'' reads in a gtf file
  \item ``rda'' load an RData object. The object needs to be named
    \Robject{gAnnot} and of class \Rclass{RangedData}.
\end{itemize}

The reads can be read in from BAM files or any format supported by \Rpackage{ShortRead}.

The reads can be summarized by:
\begin{itemize}
  \item exons 
  \item features (any features such as introns, enhancers, \emph{etc.})
  \item transcripts
  \item geneModels (a geneModel is the set of non overlapping loci
    (\emph{i.e.} synthetic exons) that represents all the possible
    exons and UTRs of a gene. Such geneModels are essential when
    counting reads as they ensure that no reads will be accounted for
    several times. \emph{E.g.}, a gene can have different isoforms,
    using different exons, overlapping exons, in which
    case summarizing by exons might result in counting a read several
    times, once per overlapping exon. \emph{N.B.} Assessing differential
    expression between transcripts, based on synthetic exons is
    something possible since the release \emph{2.14} of R, using the
    \Rpackage{DEXSeq} package available from Bioconductor.
\end{itemize}

The results can be exported in four different formats:
\begin{itemize}
\item count table (the default, a n (features) x  m (samples) \Robject{matrix}).
\item a \Rpackage{DESeq} \citep{Anders:2010p1659}
  \Rclass{countDataSet} class object. Useful to perform further
  analyses using the \Rpackage{DESeq} package.
\item an \Rpackage{edgeR} \citep{Robinson:2010p775} \Rclass{DGEList}
  class object. Useful to perform further analyses using the
  \Rpackage{edgeR} package.
\item an \Rclass{RNAseq} class object. Useful for performing
  additional pre-processing without re-loading the reads and
  annotations.
\end{itemize}

For more details and a complete overview of the \Rpackage{easyRNASeq}
package capabilities, have a look at the \Rpackage{easyRNASeq} vignette.

<<open the vignette, eval=FALSE>>=
vignette("easyRNASeq")
@ 

The obtained results can optionally be normalized as \emph{Reads per Kilobase
  of feature per Million reads in the library} (RPKM,
\cite{Mortazavi:2008p740}) or using the \Rpackage{DESeq} or
\Rpackage{edgeR} packages. 
\newline
\newline
\textbf{Q12}: From the same input files and annotations, generate an object of
class \Rclass{RNAseq} .
\label{question12}
\newline
\textbf{Q13}: Summarize the counts per transcript and geneModels.
\label{question13}
\newpage

\paragraph{Finally...}
If you find \Rpackage{easyRNASeq} useful and apply it in the
frame of your research for a publication, please cite it:

\subparagraph{}
easyRNASeq: a bioconductor package for processing RNA-Seq data\\
Nicolas Delhomme; Ismael Padioleau; Eileen E. Furlong; Lars M. Steinmetz\\
Bioinformatics 2012; doi: 10.1093/bioinformatics/bts477\\

\section{Advanced usage}

In this section we will discuss about more advanced RNA-Seq
pre-processing, such as de-multiplexing, normalizing or
\emph{de novo} identification of expressed regions.

\subsection{Normalizing counts}

A common way to normalize reads is to convert them to RPKM. This implies
normalizing the read counts depending on the genic feature size (exon,
transcript, gene model,...) and on the total number of reads sequenced
for that library. \Rpackage{easyRNASeq} count tables can be easily
transformed into RPKM, by using the \Rfunction{RPKM} method:

<<RPKM counts, eval=FALSE>>=
feature.size = width(exon.range2)
names(feature.size) = exon.range2$exon
feature.size <- feature.size[!duplicated(names(feature.size))]
lib.size=c("ACACTG.bam"=56643,
  "ACTAGC.bam"=42698,
  "ATGGCT.bam"=55414,
  "TTGCGA.bam"=60740)
head(RPKM(count.table,NULL,
          lib.size=lib.size,
          feature.size=feature.size))
@

\textbf{Q14}: Do the same for the object created at the \textbf{Q12}
and \textbf{Q13} for every possible counts. 
\label{question14}
\newline
\newline
Such a count normalization is suited for visualization, but
sub-optimal for further analyses . A better way of normalizing the
data is to use either the \Rpackage{edgeR} or \Rpackage{DESeq}
packages, provided you have got enough (biological) replicates. Refer
to the \Rpackage{easyRNASeq}, the \Rpackage{DESeq} and the
\Rpackage{edgeR} vignettes. 


\textbf{Q15}: Perform the examples in the \Rpackage{easyRNASeq} vignette paragraph 3.7.

\subsection{De-multiplexing samples}

This part of the tutorial is now in the \Rpackage{easyRNASeq} vignette paragraph 4.


\textbf{Q16}: Perform the examples in the \Rpackage{easyRNASeq} vignette paragraph 4.
\newline

\textbf{Q17}: How would you access the barcode sequence in the \Robject{alns} object?
\label{question17}
\newline


\begin{comment}
  TODO Do it.
  Mention window and stuff ( see M.M. talk at the EMBL in 2011)
\end{comment}

%\subsection{De-novo identification of expressed islands}

%Visually assessing the data is important to make sure that the raw
%data you are looking at agrees with the experimental design that was
%performed. This will be the topic of the next (and last) section.
%s\newpage

\section{Visualizing the data}

Before performing any more advanced analyses, it is crucial to be able
to visualize the data. Many technical or procedural problems can be
identified and resolved, making the residual filtered data of better
quality. 
\newline
The \Rpackage{rtracklayer} library provides the necessary
functionalities to export data stored in \Rclass{GenomicData} and
\Rclass{RangedData} class objects.

\subsection{exporting the coverage}

A common technical problem in NGS data is PCR amplification
biases. These can be visualized quite easily by scanning the read
coverage across chromosomes in a \emph{Genome Browser}. To achieve
this, one needs to export the coverage into a wig formatted file. The
wig format expects constant span and constant steps. This is
problematic since, if you have a step of 50bp and want a span of 50
bp, you'd need a chromosome whose size is a multiple of 50bp. Luckily
the chromosome size is not enforced by Genome Browsers, so a small
hack (5th line) does it (actually it is useless here as the chromosome
4 size is a natural multiple of the selected window size). 

<<Chromosome 4 wig export, eval=FALSE>>=
library(rtracklayer)
window.size <- 51
rngs <- breakInChunks(length(cover[["chr4"]]),window.size)
vals <- viewSums(Views(cover[["chr4"]],rngs))
width(rngs)[width(rngs) != width(rngs)[1]] <- width(rngs)[1]
silent <- export(
                 RangedData(rngs,score=vals,universe="Dmelanogaster",space="chr4"),
                 con="chr4.wig"
                 )
@ 

\subsection{exporting the normalized exon counts}

Depending on the kind of experiments, other criteria can be visually
assessed. For example, in the case of a gene over-expression in a
sample, one could visually check the score obtained for that gene.

<< normalized exon bed export, eval=FALSE>>=
exon.RPKM <- easyRNASeq(system.file(
                                    "extdata",
                                    package="RnaSeqTutorial"),
                        organism="Dmelanogaster",
                        chr.sizes=as.list(seqlengths(Dmelanogaster)),
                        readLength=36L,
                        annotationMethod="rda",
                        annotationFile=system.file(
                          "data",
                          "gAnnot.rda",
                          package="RnaSeqTutorial"),
                        format="aln",
                        count="exons",
                        normalize=TRUE,
                        pattern="subset_export",
                        type="SolexaExport",
                        filter=compose(
                          chastityFilter(),
                          nFilter(2),
                          chromosomeFilter(regex="chr")))

exons <- exon.range2
exons <- exons[!duplicated(exons$exon),]
exons$score <- exon.RPKM[,1]
exons$name <- rownames(exon.RPKM)
exons <- exons[exons$score>0,]
export(exons,con="exons.bed")
@ 

\subsection{conclusion}

In this section, we have seen how to export the data in a lightweight
fashion to be visualized in a Genome Browser. This step is an
\textbf{essential} step for validating the data. The QA processes run
on the raw or aligned data might reveal \textit{technical} issues,
but other biases might still be present in your data and the best
(only?) way to control for those is visual. For example, one could
compare replicates (in which case, biological replicates
are best), by plotting a scatterplot of both replicates and
estimating their correlation. Keeping in mind the design of the
experiments, helps design the necessary QA step one can do;
\emph{i.e.} In an RNA-Seq experiment, where a gene has been
over-expressed, you would expect to be able to visualize it when
comparing it to a control sample.

\newpage
\section{You are done, but still there is more to come...}

<<final.cleanup, eval=TRUE,echo=FALSE>>=
file2clean=c(
  "chr4.wig",
  "Rplots.pdf",
  "subset.bam",
  "subset.bam.bai")
silent <- sapply(
                 file2clean,
                 function(f2c){
                   if(file.exists(f2c)){file.remove(f2c)}
                 })
@ 

New protocols, new packages are constantly being developed; making
pre-processing NGS data a moving target. For example, it is known that
the standard Illumina RNA-Seq protocol shows a bias in the first 12
nucleotides of every read. It is still unclear where this bias comes
from (fragmentation, random hexamer priming, RNAseH sequence
specificity), but there has been a couple of publication recently that
proposes corrections for that bias \citep{Li:2010p732, Hansen:2010p495}.
\newline
\newline
Feedback and requests are very welcome. Just
look at the \emph{Final Remarks section} \ref{sec:finalRem},
page \pageref{sec:finalRem}. for contact details.

\newpage

\section{Session Information}

The version number of R\citep{ref:R} and packages loaded for generating the vignette were:

<<SessionInfo, eval=TRUE, echo=FALSE>>=
##library(rtracklayer)
library(BSgenome.Dmelanogaster.UCSC.dm3)
library(easyRNASeq)
sessionInfo()
@

\newpage

\section{Final remarks}
\label{sec:finalRem}
RNA-Seq is still maturating and a lot of new developments are to be
expected. If you have any questions, comments, feel free to contact me:
delhomme \emph{at} embl \emph{dot} de.
\newline
The author want to thank \emph{Gabriella Rustici} for her time,
comments and patience, as well as for organizing the course.


\newpage

\bibliographystyle{plainnat}

\bibliography{RnaSeqTutorial}

\newpage

\appendix
\section{Appendix A: Solutions}
\label{apdxA}

\begin{itemize}

\item \textbf{Q1}: section \ref{question1}, page \pageref{question1}: The \Robject{aln} and
\Robject{aln2} objects differs by their number of reads. In the
\Robject{aln2}, the \emph{chastity filtered} reads have been ignored.

\item \textbf{Q2}: section \ref{question2}, page \pageref{question2}: The \Robject{aln3}, of class
\Rclass{GAlignments} from the \Rpackage{GenomicRanges} package
does not contain any sequence, quality or id information.

\item \textbf{Q3}: section \ref{question3}, page \pageref{question3}:
using the \Rfunction{ngap} function gives you the number of
alignment having a gap, but not the size of that gap. This can be
accessed using the \Rfunction{cigarOpTable} and \Rfunction{cigar} as
in the example below: 
  
<<solution 3, eval=FALSE>>=
table(ngap(aln3))
table(cigarOpTable(cigar(aln3))[,"N"])
@ 

\item \textbf{Q4}: section \ref{question4}, page \pageref{question4}: using the \Rfunction{GRanges}
constructor as follow:
<<solution 4, eval=FALSE>>=
exon.grange <- GRanges(
                      IRanges(
                              start=exon.annotation$exon_chrom_start,
                              end=exon.annotation$exon_chrom_end),
                      seqnames=Rle(exon.annotation$chromosome),
                      strand=Rle(exon.annotation$strand),
                      transcript=exon.annotation$ensembl_transcript_id,
                      gene=exon.annotation$ensembl_gene_id,
                      exon=exon.annotation$ensembl_exon_id,
                      seqlengths = chrSizes[
                        match(
                              unique(exon.annotation$chromosome),
                              names(chrSizes))]
                       )
@ 

\item \textbf{Q5}: section \ref{question5}, page \pageref{question5}: using the \Rfunction{GRanges}
constructor as follow:
<<solution 5, eval=FALSE>>=
levels(gInterval$strand) <- c("-","+")
exon.grange2 <- GRanges(
                        IRanges(
                                start=gInterval[,1],
                                end=gInterval[,2]),
                        seqnames=Rle(gInterval$seq_name),
                        strand=Rle(gInterval$strand),
                        transcript=as.vector(getGffAttribute(
                          gInterval,"Parent")),
                        gene=as.vector(getGffAttribute(
                          gInterval,"Name")),
                        exon=as.vector(getGffAttribute(
                          gInterval,"ID")),
                        seqlengths = chrSizes[
                          match(
                                unique(gInterval$seq_name),
                                names(chrSizes))]
                          )
@ 

\item \textbf{Q6}: section \ref{question6}, page \pageref{question6}: using the
\Rpackage{rtracklayer} package, specifically the
\Rfunction{export.gff3} of \Rfunction{export.gff} or the
\Rfunction{export} function as follow. The only difference in using these
functions is that the number of argument you need to provide somewhat increases
as the function becomes more generic. The three following examples
have the same results.
<<solution 6, eval=FALSE>>=
library(rtracklayer)
load(system.file("data",
                 "gAnnot.rda",
                 package="RnaSeqTutorial"))
export.gff3(gAnnot,con="annot.gff")
export.gff(gAnnot,con="annot.gff",version="3")
export(gAnnot,con="annot.gff",version="3")
@ 

\item \textbf{Q7}: section \ref{question7}, page \pageref{question7}: simply coerce the
\Rclass{Rle} class object into an integer.

<<solution7, eval=FALSE>>=
as.integer(cover$chr4) 
@


\item \textbf{Q8}: section \ref{question8}, page \pageref{question8}: use the \Rfunction{runLength}
and \Rfunction{runValue} functions

<<solution8, eval=FALSE>>=
runLength(cover$chr4)
runValue(cover$chr4)
@

\item \textbf{Q9}: section \ref{question9}, page \pageref{question9}: The names are different for
both objects. It is therefore essential to make sure that there are
ordered in the same way to avoid unexpected results. In standard R,
names are optional for lists; that is the default behavior, so do not
expect otherwise from packages until you've tested it. Better safe
than sorry.

<<solution9 better safe than sorry, eval=FALSE>>=
names(exon.range2)
names(cover)
match(names(exon.range2),names(cover))
@ 

\item \textbf{Q10}: section \ref{question10}, page \pageref{question10}: The main drawback is an
edge effect; \emph{i.e.} reads that are spanning the exon boundaries,
although valid will not be taken entirely into account, only the
proportion of these reads that cover the exon will. In addition, as
shown below, exons present in different isoforms will be several times
accounted for.


<<solution10, eval=FALSE>>=
exon.coverage <- unlist(exon.coverage)
names(exon.coverage) <- exon.range2$exon
head(
     sort(
          exon.coverage[!duplicated(names(exon.coverage))],
          decreasing=TRUE))
@

\item \textbf{Q11}: section \ref{question11}, page \pageref{question11}:
<<solution11,eval=FALSE>>=
sel <- chromosome(aln) != "chrM"
aln <- aln[sel]
exon.counts <- countOverlaps(
                             exon.range2,
                             split(IRanges(
                                           start=position(aln),
                                           width=width(aln)),
                                   chromosome(aln))
                             )
@ 

We might want to compare that result with the former one, stored in
the \Robject{exon.coverage}.
<<solution11.2,eval=FALSE>>=

plot(
     unlist(exon.coverage),
     unlist(exon.counts),
     log="xy",
     main="countOverlap vs. aggregate",
     xlab="aggregate",
     ylab="CountOverlap",
     pch="+",col=6)
abline(0,1,lty=2,col="orange")

table(unlist(exon.coverage) - unlist(exon.counts))
@ 

As you can see, the difference is not striking.

\item \textbf{Q12}: section \ref{question12}, page \pageref{question12}: as in the example, but
add the \Rfunction{outputFormat} argument as follow:

<<solution12,eval=FALSE>>=
rnaSeq <- easyRNASeq(system.file(
                                 "extdata",
                                 package="RnaSeqTutorial"),
                     organism="Dmelanogaster",
                     chr.sizes=as.list(seqlengths(Dmelanogaster)),
                     readLength=36L,
                     annotationMethod="rda",
                     annotationFile=system.file(
                       "data",
                       "gAnnot.rda",
                       package="RnaSeqTutorial"),
                     format="bam",
                     count="exons",
                     pattern="bam$",
                     outputFormat="RNAseq")
show(rnaSeq)
@ 

\item \textbf{Q13}: section \ref{question13}, page \pageref{question13}: use the
\Rfunction{transcriptCounts}, \Rfunction{geneCounts} to generate the
counts and \Rfunction{readCounts} to access the results.

<<Transcript counts, eval=FALSE>>=
rnaSeq <- transcriptCounts(rnaSeq)
head(readCounts(rnaSeq,'transcripts'))
@ 

Summarizing by transcript introduces some complexity in the data
analysis, \emph{i.e.} exons part of different isoforms introduce a
bias in the counts. For that reason, it might be better to have a
first look at the data, summarized by genes. This, however, requires to
combine all the alternative exons and UTRs present for every gene into
a ``gene model''; \emph{i.e.} overlapping exons are merged into
``synthetic'' ones. This is what is performed when the arguments
``count'' and ``summarization'' are set to ``genes'' and
``geneModels'', respectively. A caveat not addressed by this procedure
are genes overlapping on the same or opposite strands. If this occurs
a warning will be emitted. If the reads were summarized by
``geneModels'' and the ``outputFormat'' argument was set to
``RNAseq'', one can use the ``geneModel'' accessor on the obtained
object to access the computed gene models. They are stored in an
\Rclass{RangedData} object and can be modified to address the caveat
previously mentioned. To be strict, one would remove every overlapping
loci and conserve only the other ones. Such a modified annotation can
then be saved and used for the next \Rfunction{easyRNASeq} run.
\newline
It is not possible yet to summarize by ``geneModels'' using
the \Rfunction{geneCounts}  function. A meaningful error
message is thrown if the \Rfunction{geneCounts}  is used for that
purpose.

<<solution13, eval=FALSE>>=
rnaSeq<-geneCounts(rnaSeq,summarization='geneModels')
@ 

This behavior will be corrected in the next release. At the moment,
this has to be done using the \Rfunction{easyRNASeq} function
directly. 

<<solution13B, eval=FALSE>>=
rnaSeq2 <- easyRNASeq(system.file(
                                  "extdata",
                                  package="RnaSeqTutorial"),
                      organism="Dmelanogaster",
                      chr.sizes=as.list(seqlengths(Dmelanogaster)),
                      readLength=36L,
                      annotationMethod="rda",
                      annotationFile=system.file(
                        "data",
                        "gAnnot.rda",
                        package="RnaSeqTutorial"),
                      format="bam",
                      count="genes",
                      summarization="geneModels",
                      pattern="bam$",
                      outputFormat="RNAseq")
head(readCounts(rnaSeq2,'genes','geneModels'))
@

\item \textbf{Q14}: section \ref{question14}, page \pageref{question14}: you can directly use the
\Robject{rnaSeq} and \Robject{rnaSeq2} objects
<<solution14,eval=FALSE>>=
RPKM(rnaSeq,from="transcripts")
RPKM(rnaSeq2,from="geneModels")
@ 

\item \textbf{Q15}: Explanations are in the \Rpackage{easyRNASeq}
  package vignette.
  
\item \textbf{Q16}: Explanations are in the \Rpackage{easyRNASeq}
  package vignette.

\item \textbf{Q17}: section \ref{question17}, page \pageref{question17}: For the Illumina protocol,
the barcode is read in a separate sequencing reaction. The barcode
sequence is reported as a field of the \emph{export} file, and when
the data is loaded using the\Rfunction{withAll} argument, it is
accessible through:

<<solution17,eval=FALSE>>=
alignData(alns)$multiplexIndex
@ 

To view all the possible fields, do:
<<solution17b, eval=FALSE>>=
varLabels(alignData(alns))
@ 

\end{itemize}

\end{document}



% \subsection{\emph{De novo} transcript identification}

% TODO Move that to appendix B
% TODO and fix it

% RNAseq experiments are very interesting in the sense that all
% transcribed molecule can be identified, as the technique is not
% dependent on a predefined sets of probes, as micro-arrays
% are. Therefore unknown transcripts and isoforms, as well as regulatory
% transcribed elements can be readily identified from RNAseq data. To
% that extend, several methods are available to recreate and annotate
% transcripts like Oases, Velvet
% \citep{Zerbino:2008p749,Zerbino:2009p743},
% tophat\citep{Trapnell:2009p156} to cite some of them (see
% \citep{Ameur:2010p280} as well) but few has been done for other
% regulatory transcribed elements like eRNAs \citep{Kim:2010p572}. For
% that reason, we have introduced in this package a very basic approach
% to identify locus and quantify counts without prior annotation
% knowledge. It searches for \emph{islands} where the coverage is higher
% than the \emph{background}, for a long enough distance, still allowing
% for local gaps.

% <<% Island calls, eval=TRUE>>=
% % islandCounts(rnaSeq,max.gap=35,min.length=140,min.cov=4L)
% % % look at the island annotations
% % readIslands(rnaSeq)
% % % look at the count
% % islands <- readCounts(rnaSeq,'islands')
% % % find the loci of the most expressed island
% % big.island <- readIslands(rnaSeq)[
% % 	readIslands(rnaSeq)$names %in% 
% % 	names(sort(islands[[1]],decreasing=TRUE)[1]),]
% % % what is the island size?
% % width(big.island)
% % start(big.island)
% % @ 

% This locus is occupied by the gene \emph{MCPH1}, putatively involved
% in development, fact that fits with the nature of the sample extracted
% from drosophila embryos. 

% All together, this is still work in progress and functionalities still
% need to be added, in particular when dealing with multiple samples,
% \emph{i.e.} how to combine the different islands identified by different
% samples. An approach similar to the one used to generate the geneModel
% could be a solution, for example.

% \begin{comment}
% Lab should only present material that you are reasonably sure of.
% \end{comment}

% @article{Ameur:2010p280,
% author = {Adam Ameur and Anna Wetterbom and Lars Feuk and Ulf Gyllensten}, 
% journal = {Genome Biology 2010 11:202},
% title = {Global and unbiased detection of splice junctions from RNA-Seq data},
% abstract = {ABSTRACT: We have developed a new strategy for de novo prediction of splice junctions in short read RNA-Seq data, suitable for detection of novel splicing events and chimeric transcripts. When tested on mouse RNA-Seq data, over 31,000 splice events were predicted, of which 88% bridged between two regions separated by at most 100 kb, and 74% connected two exons of the same RefSeq gene. Our method also reports genomic rearrangements such as insertions and deletions.},
% number = {3},
% pages = {R34},
% volume = {11},
% year = {2010},
% month = {Mar},
% }

% @article{Zerbino:2008p749,
% author = {Daniel R Zerbino and Ewan Birney}, 
% journal = {Genome Research},
% title = {Velvet: algorithms for de novo short read assembly using de Bruijn graphs},
% abstract = {We have developed a new set of algorithms, collectively called "Velvet," to manipulate de Bruijn graphs for genomic sequence assembly. A de Bruijn graph is a compact representation based on short words (k-mers) that is ideal for high coverage, very short read (25-50 bp) data sets. Applying Velvet to very short reads and paired-ends information only, one can produce contigs of significant length, up to 50-kb N50 length in simulations of prokaryotic data and 3-kb N50 on simulated mammalian BACs. When applied to real Solexa data sets without read pairs, Velvet generated contigs of approximately 8 kb in a prokaryote and 2 kb in a mammalian BAC, in close agreement with our simulated results without read-pair information. Velvet represents a new approach to assembly that can leverage very short reads in combination with read pairs to produce useful assemblies.},
% affiliation = {EMBL-European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom.},
% number = {5},
% pages = {821--9},
% volume = {18},
% year = {2008},
% month = {May},
% }