<tool id="diffbind" name="DiffBind" version="2.6.5.0">
    <description> differential binding analysis of ChIP-Seq peak data</description>
    <requirements>
        <requirement type="package" version="2.6.5">bioconductor-diffbind</requirement>
        <requirement type="package" version="1.20.0">r-getopt</requirement>
        <!--added rmysql requirement to remove: "Warning: namespace ‘RMySQL’ is not available"-->
        <requirement type="package" version="0.10.11">r-rmysql</requirement>
    </requirements>
    <stdio>
        <regex match="Execution halted"
           source="both"
           level="fatal"
           description="Execution halted." />
        <regex match="Input-Error 01"
           source="both"
           level="fatal"
           description="Error in your input parameters: Make sure you only apply factors to selected samples." />
        <regex match="Error in"
           source="both"
           level="fatal"
           description="An undefined error occured, please check your intput carefully and contact your administrator." />
    </stdio>
    <version_command><![CDATA[
echo $(R --version | grep version | grep -v GNU)", DiffBind version" $(R --vanilla --slave -e "library(DiffBind); cat(sessionInfo()\$otherPkgs\$DiffBind\$Version)" 2> /dev/null | grep -v -i "WARNING: ")," getopt version" $(R --vanilla --slave -e "library(getopt); cat(sessionInfo()\$otherPkgs\$getopt\$Version)" 2> /dev/null | grep -v -i "WARNING: ")", rmysql version" $(R --vanilla --slave -e "library(rmysql); cat(sessionInfo()\$otherPkgs\$rmysql\$Version)" 2> /dev/null | grep -v -i "WARNING: ")
    ]]></version_command>
    <command><![CDATA[
        ## seems that diffbind also needs file extensions to work properly
        #set $counter = 1
        #for $sample in $samples:
            ln -s $sample.bamreads #echo str($counter) + "_bamreads.bam"# &&
            ln -s ${sample.bamreads.metadata.bam_index} #echo str($counter) + "_bamreads.bai"# &&
            #if str( $sample.bamcontrol ) != 'None':
                ln -s $sample.bamcontrol #echo str($counter) + "_bamcontrol.bam"# &&
                ln -s ${sample.bamcontrol.metadata.bam_index} #echo str($counter) + "_bamcontrol.bai"# &&
            #end if
            #set $counter = $counter + 1
        #end for

        Rscript '$__tool_directory__/diffbind.R'
            -i $infile
            -o '$outfile'
            -p '$plots'
            -f $format
            -t $th

            #if $binding_affinity_matrix:
                -b
            #end if
]]>
    </command>
    <configfiles>
<configfile name="infile"><![CDATA[
#set $counter = 1
#for $sample in $samples:
#if str( $sample.bamcontrol ) != 'None' and $counter == 1:
SampleID,Tissue,Factor,Condition,Replicate,bamReads,bamControl,Peaks
#elif $counter == 1:
SampleID,Tissue,Factor,Condition,Replicate,bamReads,Peaks
#end if
#if str( $sample.bamcontrol ) != 'None':
$sample.sample_id,$sample.tissue,$sample.factor,$sample.condition,$sample.replicate,#echo str($counter) + '_bamreads.bam'#,#echo str($counter) + '_bamcontrol.bam'#,$sample.peaks
#else:
$sample.sample_id,$sample.tissue,$sample.factor,$sample.condition,$sample.replicate,#echo str($counter) + '_bamreads.bam'#,$sample.peaks
#end if
#set $counter = $counter + 1
#end for]]></configfile>
    </configfiles>
    <inputs>
        <repeat name="samples" title="Samples" min="2">
            <param name="sample_id" type="text" value="Sample ID" label="Specify a sample id" help="e.g. BT474.1-" />
            <param name="tissue" type="text" value="Tissue" label="Specify the tissue" help="e.g. BT474" />
            <param name="factor" type="text" value="Factor Name" label="Specify a factor name" help="e.g. ER" />
            <param name="condition" type="text" value="Condition" label="Specify the condition" help="e.g. Resistent" />
            <param name="replicate" type="integer" value="1" label="Specify the replicate number" help="e.g. 1" />
            <param name="bamreads" type="data" format="bam" label="Read BAM file" help="Specify the Read BAM file, used for Peak calling."/>
            <param name="bamcontrol" type="data" format="bam" optional="True" label="Control BAM file" help="If specifying a control BAM file for this sample, then all samples are required to specify one."/>
            <param name="peaks" type="data" format="bed" label="Peak file" help="Result of your Peak calling experiment."/>
        </repeat>
        <param name="th" type="float" value="1" min="0" max="1"
                label="FDR Threshold"
                help="Significance threshold; all sites with FDR less than or equal to this value will be included in the report. A value of 1 will include all binding sites in the report. Default: 1"/>
        <param name="pdf" type="boolean" truevalue="" falsevalue="" checked="true"
            label="Visualising the analysis results"
            help="output an additional PDF file" />
        <param name="format" type="select" label="Output Format">
            <option value="bed">BED</option>
            <option value="gff">GFF</option>
            <option value="wig">WIG</option>
        </param>
        <param name="binding_affinity_matrix" type="boolean" truevalue="True" falsevalue="" checked="False" label="Output binding affinity matrix?" help="Output a table of the binding scores" />
    </inputs>
    <outputs>
        <data name="outfile" format="bed" label="Differential binding sites on ${on_string}">
            <change_format>
                <when input="format" value="wig" format="wig" />
                <when input="format" value="gff" format="gff" />
            </change_format>
        </data>
        <data name="plots" format="pdf" label="Differential binding sites on ${on_string}">
            <filter>pdf == True</filter>
        </data>
        <data name="binding_matrix" format="tabular" from_work_dir="bmatrix.tab" label="Differential binding sites on ${on_string}">
            <filter>binding_affinity_matrix == True</filter>
        </data>
    </outputs>
    <tests>
        <test>
            <repeat name="samples">
                <param name="sample_id" value="BT4741" />
                <param name="tissue" value="BT474" />
                <param name="factor" value="ER" />
                <param name="condition" value="Resistant" />
                <param name="replicate" value="1" />
                <param name="bamreads" ftype="bam" value="BT474_ER_1.bam" />
                <param name="peaks" ftype="bed" value="BT474_ER_1.bed.gz" />
            </repeat>
            <repeat name="samples">
                <param name="sample_id" value="BT4742" />
                <param name="tissue" value="BT474" />
                <param name="factor" value="ER" />
                <param name="condition" value="Resistant" />
                <param name="replicate" value="2" />
                <param name="bamreads" ftype="bam" value="BT474_ER_2.bam" />
                <param name="peaks" ftype="bed" value="BT474_ER_2.bed.gz" />
            </repeat>
            <repeat name="samples">
                <param name="sample_id" value="MCF71" />
                <param name="tissue" value="MCF7" />
                <param name="factor" value="ER" />
                <param name="condition" value="Responsive" />
                <param name="replicate" value="1" />
                <param name="bamreads" ftype="bam" value="MCF7_ER_1.bam" />
                <param name="peaks" ftype="bed" value="MCF7_ER_1.bed.gz" />
            </repeat>
            <repeat name="samples">
                <param name="sample_id" value="MCF72" />
                <param name="tissue" value="MCF7" />
                <param name="factor" value="ER" />
                <param name="condition" value="Responsive"  />
                <param name="replicate" value="2" />
                <param name="bamreads" ftype="bam" value="MCF7_ER_2.bam" />
                <param name="peaks" ftype="bed" value="MCF7_ER_2.bed.gz" />
            </repeat>
            <param name="pdf" value="True" />
            <param name="binding_affinity_matrix" value="True" />
            <output name="outfile" value="out_diffbind.bed" />
            <output name="binding_matrix" value="out_binding.matrix" />
        </test>
    </tests>
    <help><![CDATA[

.. class:: infomark

**What it does**

DiffBind_ is a `Bioconductor package`_ that provides functions for processing ChIP-Seq data enriched for genomic loci where specific
protein/DNA binding occurs, including peak sets identified by ChIP-Seq peak callers and
aligned sequence read datasets. It is designed to work with multiple peak sets simultaneously,
representing different ChIP experiments (antibodies, transcription factor and/or histone
marks, experimental conditions, replicates) as well as managing the results of multiple peak
callers.

The primary emphasis of DiffBind is on identifying sites that are differentially bound
between two sample groups. It includes functions to support the processing of peak sets,
including overlapping and merging peak sets, counting sequencing reads overlapping intervals
in peak sets, and identifying statistically significantly differentially bound sites based on
evidence of binding affinity (measured by differences in read densities). To this end it uses
statistical routines developed in an RNA-Seq context (primarily the Bioconductor packages
edgeR and DESeq2 ). Additionally, the package builds on Rgraphics routines to provide a
set of standardized plots to aid in binding analysis.

The `DiffBind User Guide`_ includes a brief overview of the processing flow, followed by four sections of
examples: the first focusing on the core task of obtaining differentially bound sites based on
affinity data, the second working through the main plotting routines, the third discussing the
use of a blocking factor, and the fourth revisiting occupancy data (peak calls) in more detail,
as well as comparing the results of an occupancy-based analysis with an affinity-based one.
Finally, certain technical aspects of the how these analyses are accomplished are detailed.

Note DiffBind requires a minimum of four samples (two groups with two replicates each).

.. _DiffBind: https://bioconductor.org/packages/release/bioc/html/DiffBind.html
.. _`Bioconductor package`: https://bioconductor.org/packages/release/bioc/html/DiffBind.html
.. _`DiffBind User Guide`: https://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf

**Inputs**

DiffBind works primarily with peaksets, which are sets of genomic intervals representing
candidate protein binding sites. Each interval consists of a chromosome, a start and end
position, and usually a score of some type indicating confidence in, or strength of, the peak.
Associated with each peakset are metadata relating to the experiment from which the peakset
was derived. Additionally, files containing mapped sequencing reads (generally .bam files) can
be associated with each peakset (one for the ChIP data, and optionally another representing
a control sample)

**Sample Information**

You have to specify your sample information in the tool form above.

Example:

    ============= ========== ========== ============= =============
     **SampleID** **Tissue** **Factor** **Condition** **Replicate**
    ------------- ---------- ---------- ------------- -------------
    BT4741        BT474      ER         Resistant     1            
    BT4742        BT474      ER         Resistant     2            
    MCF71         MCF7       ER         Responsive    1            
    MCF72         MCF7       ER         Responsive    2            
    MCF73         MCF7       ER         Responsive    3            
    T47D1         T47D       ER         Responsive    1            
    T47D2         T47D       ER         Responsive    2            
    MCF7r1        MCF7       ER         Resistant     1            
    MCF7r2        MCF7       ER         Resistant     2            
    ZR751         ZR75       ER         Responsive    1            
    ZR752         ZR75       ER         Responsive    2            
    ============= ========== ========== ============= =============

Or provide a sample sheet tabular file such as below.

Example:

    ======== ======  ====== ========== ========== ========= ====================  ========= ===================== ================= ==========
    SampleID Tissue  Factor Condition  Treatment  Replicate bamReads              ControlID bamControl            Peaks             PeakCaller
    ======== ======  ====== ========== ========== ========= ====================  ========= ===================== ================= ==========
    BT4741   BT474   ER     Resistant  Full-Media  1        Chr18_BT474_ER_1.bam  BT474c    Chr18_BT474_input.bam BT474_ER_1.bed.gz bed
    BT4742   BT474   ER     Resistant  Full-Media  2        Chr18_BT474_ER_2.bam  BT474c    Chr18_BT474_input.bam BT474_ER_2.bed.gz bed
    MCF71    MCF7    ER     Responsive Full-Media  1        Chr18_MCF7_ER_1.bam   MCF7c     Chr18_MCF7_input.bam  MCF7_ER_1.bed.gz  bed
    MCF72    MCF7    ER     Responsive Full-Media  2        Chr18_MCF7_ER_2.bam   MCF7c     Chr18_MCF7_input.bam  MCF7_ER_2.bed.gz  bed
    MCF73    MCF7    ER     Responsive Full-Media  3        Chr18_MCF7_ER_3.bam   MCF7c     Chr18_MCF7_input.bam  MCF7_ER_3.bed.gz  bed
    T47D1    T47D    ER     Responsive Full-Media  1        Chr18_T47D_ER_1.bam   T47Dc     Chr18_T47D_input.bam  T47D_ER_1.bed.gz  bed
    T47D2    T47D    ER     Responsive Full-Media  2        Chr18_T47D_ER_2.bam   T47Dc     Chr18_T47D_input.bam  T47D_ER_2.bed.gz  bed
    MCF7r1   MCF7    ER     Resistant  Full-Media  1        Chr18_TAMR_ER_1.bam   TAMRc     Chr18_TAMR_input.bam  TAMR_ER_1.bed.gz  bed
    MCF7r2   MCF7    ER     Resistant  Full-Media  2        Chr18_TAMR_ER_2.bam   TAMRc     Chr18_TAMR_input.bam  TAMR_ER_2.bed.gz  bed
    ZR751    ZR75    ER     Responsive Full-Media  1        Chr18_ZR75_ER_1.bam   ZR75c     Chr18_ZR75_input.bam  ZR75_ER_1.bed.gz  bed
    ZR752    ZR75    ER     Responsive Full-Media  2        Chr18_ZR75_ER_2.bam   ZR75c     Chr18_ZR75_input.bam  ZR75_ER_2.bed.gz  bed
    ======== ======  ====== ========== ========== ========= ====================  ========= ===================== ================= ==========


**Peak files**

Result of your Peak calling experiment in bed format, one file for each sample is required.

Example:

    ======= ======= ======= =============== =======
    1          2      3          4           **5**
    ======= ======= ======= =============== =======
    chr18   215562  216063  MACS_peak_16037 56.11
    chr18   311530  312105  MACS_peak_16038 222.49
    chr18   356656  357315  MACS_peak_16039 92.06
    chr18   371110  372092  MACS_peak_16040 123.86
    chr18   395116  396464  MACS_peak_16041 1545.39
    chr18   399014  400382  MACS_peak_16042 1835.19
    chr18   499134  500200  MACS_peak_16043 748.32
    chr18   503518  504552  MACS_peak_16044 818.30
    chr18   531672  532274  MACS_peak_16045 159.30
    chr18   568326  569282  MACS_peak_16046 601.11
    ======= ======= ======= =============== =======

* BAM file which contains the mapped sequencing reads can be associated with each peakset
* Control BAM file represents a control dataset and are optional, but have to specified for all when used.


**Outputs**

As output format you can choose BED, GFF, WIG.

Example:

======== ====== =======+
seqnames ranges strand             Conc Conc_Resistant

2452     chr18 [64490686, 64491186] * | 6.36 1.39
1291     chr18 [34597713, 34598213] * | 5.33 0.22
976      chr18 [26860997, 26861497] * | 7.3 3.13
2338     chr18 [60892900, 60893400] * | 7.13 1.84
2077     chr18 [55569087, 55569587] * | 5.52 1.89

Conc_Responsive Fold p-value FDR
<numeric> <numeric> <numeric> <numeric>
2452 7 -5.61 3.57e-10 1.02e-06
1291 5.97 -5.75 1.1e-09 1.57e-06
976 7.92 -4.79 1.1e-08 1.05e-05
2338 7.77 -5.93 1.68e-08 1.17e-05
2077 6.13 -4.23 2.36e-08 1.17e-05

The value columns show the
Conc mean read concentration over all the samples (the default calculation uses log2 normalized ChIP read counts with control read counts subtracted) 
Conc_Resistant mean concentration over the first (Resistant) group 
Conc_Responsive mean concentration over second (Responsive) group 
Fold column shows the difference in mean concentrations between the two groups (Conc_Resistant - Conc_Responsive), with a positive value indicating increased binding affinity in the Resistant group and a negative value indicating increased binding affinity in the Responsive group.
p-value confidence measure for identifying these sites as differentially bound 
FDR a multiple testing corrected FDR p-value


**Binding Affinity Matrix**

The final result of counting is a binding affinity matrix containing a (normalized) read count for each sample at every potential binding site. With this matrix, the samples can be re-clustered using affinity, rather than occupancy, data. The binding affinity matrix can be used for QC plotting as well as for subsequent
differential analysis.

Example:

    ====== ====== ====== ========== ========== ========= ====== ========= ====
    ID     Tissue Factor Condition  Treatment  Replicate Caller Intervals FRiP
    ====== ====== ====== ========== ========== ========= ====== ========= ====
    BT4741 BT474  ER     Resistant  Full-Media 1         counts 2845      0.16
    BT4742 BT474  ER     Resistant  Full-Media 2         counts 2845      0.15
    MCF71  MCF7   ER     Responsive Full-Media 1         counts 2845      0.27
    MCF72  MCF7   ER     Responsive Full-Media 2         counts 2845      0.17
    MCF73  MCF7   ER     Responsive Full-Media 3         counts 2845      0.23
    T47D1  T47D   ER     Responsive Full-Media 1         counts 2845      0.10
    T47D2  T47D   ER     Responsive Full-Media 2         counts 2845      0.06
    MCF7r1 MCF7   ER     Resistant  Full-Media 1         counts 2845      0.20
    MCF7r2 MCF7   ER     Resistant  Full-Media 2         counts 2845      0.13
    ZR751  ZR75   ER     Responsive Full-Media 1         counts 2845      0.32
    ZR752  ZR75   ER     Responsive Full-Media 2         counts 2845      0.22
    ====== ====== ====== ========== ========== ========= ====== ========= ====



**More Information**

Generally, processing data with DiffBind involves five phases:

 #. Reading in peaksets
 #. Occupancy analysis
 #. Counting reads
 #. Differential binding affinity analysis
 #. Plotting and reporting


 * **Reading in peaksets**: 

The first step is to read in a set of peaksets and associated
metadata. Peaksets are derived either from ChIP-Seq peak callers, such as MACS
([1]), or using some other criterion (e.g. genomic windows, or all the promoter regions
in a genome). The easiest way to read in peaksets is using a comma-separated value
(csv) sample sheet with one line for each peakset. (Spreadsheets in Excel® format, with
a .xls or .xlsx suffix, are also accepted.) A single experiment can have more than
one associated peakset; e.g. if multiple peak callers are used for comparison purposes
each sample would have more than one line in the sample sheet. Once the peaksets
are read in, a merging function finds all overlapping peaks and derives a single set of
unique genomic intervals covering all the supplied peaks (a consensus peakset for the
experiment).

 * **Occupancy analysis**: 

Peaksets, especially those generated by peak callers, provide
an insight into the potential occupancy of the protein being ChIPed for at specific
genomic loci. After the peaksets have been loaded, it can be useful to perform some
exploratory plotting to determine how these occupancy maps agree with each other,
e.g. between experimental replicates (re-doing the ChIP under the same conditions),
between different peak callers on the same experiment, and within groups of samples
representing a common experimental condition. DiffBind provides functions to enable
overlaps to be examined, as well as functions to determine how well similar samples
cluster together. Beyond quality control, the product of an occupancy analysis may be
a consensus peakset, representing an overall set of candidate binding sites to be used
in further analysis.

 * **Counting reads**: 

Once a consensus peakset has been derived, DiffBind can use the
supplied sequence read files to count how many reads overlap each interval for each
unique sample. The peaks in the consensus peakset may be re-centered and trimmed
based on calculating their summits (point of greatest read overlap) in order to provide
more standardized peak intervals. The final result of counting is a binding affinity matrix
containing a (normalized) read count for each sample at every potential binding site.
With this matrix, the samples can be re-clustered using affinity, rather than occupancy,
data. The binding affinity matrix is used for QC plotting as well as for subsequent
differential analysis.

 * **Differential binding affinity analysis**: 

The core functionality of DiffBind is the
differential binding affinity analysis, which enables binding sites to be identified that
are statistically significantly differentially bound between sample groups. To accomplish
this, first a contrast (or contrasts) is established, dividing the samples into groups to
be compared. Next the core analysis routines are executed, by default using DESeq2 .
This will assign a p-value and FDR to each candidate binding site indicating confidence
that they are differentially bound.

 * **Plotting and reporting**: 

Once one or more contrasts have been run, DiffBind provides
a number of functions for reporting and plotting the results. MA plots give an
overview of the results of the analysis, while correlation heatmaps and PCA plots show
how the groups cluster based on differentially bound sites. Boxplots show the distribution
of reads within differentially bound sites corresponding to whether they gain or
lose affinity between the two sample groups. A reporting mechanism enables differentially
bound sites to be extracted for further processing, such as annotation, motif, and
pathway analyses.

**References**

DiffBind Authors:  Rory Stark, Gordon Brown (2011)
Wrapper authors: Bjoern Gruening, Pavankumar Videm

]]>
    </help>
    <citations>
        <citation type="doi">doi:10.1038/nature10730</citation>
    </citations>
</tool>