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<tool id="diffbind" name="DiffBind" version="2.10.0+galaxy0">
<description> differential binding analysis of ChIP-Seq peak data</description>
<xrefs>
<xref type="bio.tools">diffbind</xref>
</xrefs>
<requirements>
<requirement type="package" version="2.10.0">bioconductor-diffbind</requirement>
<requirement type="package" version="3.5.1">r-base</requirement>
<requirement type="package" version="1.20.3">r-getopt</requirement>
<requirement type="package" version="0.2.20">r-rjson</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 input 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: ")", rjson version" $(R --vanilla --slave -e "library(rjson); cat(sessionInfo()\$otherPkgs\$rjson\$Version)" 2> /dev/null | grep -v -i "WARNING: ")
]]></version_command>
<command><![CDATA[
#import re
#import json
## Adapted from DESeq2 wrapper
#set $temp_factor_names = list()
#set $temp_factor = list()
#for $g in $rep_group:
#set $peak_files = list()
#set $bam_files = list()
#set $bam_controls = list()
#for $file in $g.peaks:
#set $file_name = str($g.groupName) + "-" + re.sub('[^\w\-]', '_', str($file.element_identifier)) + "-peaks.bed"
ln -s '${file}' '${file_name}' &&
$peak_files.append($file_name)
#end for
#for $bam in $g.bamreads:
#set $bam_name = re.sub('[^\w\-]', '_', str($bam.element_identifier))
#set $bam_file = $bam_name + "-bamreads.bam"
#set $bam_index = $bam_name + "-bamreads.bai"
ln -s '${bam}' '${bam_file}' &&
ln -s '${bam.metadata.bam_index}' '${bam_index}' &&
$bam_files.append($bam_file)
#end for
$temp_factor.append( {str($g.groupName): $peak_files} )
$temp_factor.append( {str($g.groupName): $bam_files} )
#if str( $g.bamcontrol ) != 'None':
#for $ctrl in $g.bamcontrol:
#set $ctrl_name = re.sub('[^\w\-]', '_', str($ctrl.element_identifier))
#set $ctrl_file = $ctrl_name + "-bamcontrol.bam"
#set ctrl_index = $ctrl_name + "-bamcontrol.bai"
#if $ctrl_file not in json.dumps($temp_factor):
ln -s '${ctrl}' '${ctrl_file}' &&
ln -s '${ctrl.metadata.bam_index}' '${ctrl_index}' &&
#end if
$bam_controls.append($ctrl_file)
#end for
$temp_factor.append( {str($g.groupName): $bam_controls} )
#end if
#end for
$temp_factor.reverse()
$temp_factor_names.append(["Condition", $temp_factor])
Rscript '$__tool_directory__/diffbind.R'
-i '#echo json.dumps(temp_factor_names)#'
-o '$outfile'
-t $th
-f $out.format
-p '$plots'
#if $scorecol:
-n "$scorecol"
#end if
#if $lowerbetter:
-l "$lowerbetter"
#end if
#if $summits:
-s "$summits"
#end if
#if $out.binding_matrix:
-b
#end if
#if $out.rdata:
-r
#end if
#if $out.analysis_info:
-a
#end if
#if $out.rscript:
&& cp '$__tool_directory__/diffbind.R' '$rscript'
#end if
]]>
</command>
<inputs>
<repeat name="rep_group" title="Group" min="2" max="2" default="2">
<param name="groupName" type="text" label="Name"
help="Name for the Group that the peak and BAM files belong to e.g. Resistant/Responsive (two Groups must be specified for DiffBind). NOTE: Please only use letters, numbers or underscores.">
<sanitizer>
<valid initial="string.letters,string.digits"><add value="_" /></valid>
</sanitizer>
<validator type="empty_field" />
</param>
<param name="peaks" type="data" format="bed" multiple="true" label="Peak files" help="Result of your Peak calling experiment"/>
<param name="bamreads" type="data" format="bam" multiple="true" label="Read BAM files" help="Specify the Read BAM files used in the Peak calling. The input order of the BAM files for the samples MUST match the input order of the peaks files."/>
<param name="bamcontrol" type="data" format="bam" multiple="true" optional="True" label="Control BAM files" help="If specifying a control BAM file, all samples are required to specify one, see Help section below. The input order of the BAM files for the samples MUST match the input order of the peaks files."/>
</repeat>
<param name="scorecol" type="integer" min="0" value="8" label="Score Column" help="Column in peak files that contains peak scores. Default: 8 (narrowPeak)">
<sanitizer>
<valid initial="string.digits"/>
</sanitizer>
</param>
<param name="lowerbetter" type="boolean" truevalue="True" falsevalue="" checked="False" label="Lower score is better?" help="DiffBind by default assumes that a higher score indicates a better peak, for example narrowPeaks -log10pvalue. If this is not the case, for example if the score is a p-value or FDR, set this option to Yes. Default: No" />
<param name="summits" type="integer" min="0" optional="True" label="Summits" help="Extend peaks Nbp up- and downstream of the summit. For punctate peaks it is advisable to extend (e.g. 250bp), see the DiffBind User Guide">
<sanitizer>
<valid initial="string.digits"/>
</sanitizer>
</param>
<param name="th" type="float" value="0.05" 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 output. A value of 1 will output all binding sites. Default: 0.05"/>
<!-- Output Options -->
<section name="out" expanded="false" title="Output Options">
<param name="format" type="select" label="Output Format">
<option value="interval" selected="True">Interval</option>
<option value="bed">BED</option>
<option value="tabular">Tabular (tab-separated)</option>
</param>
<param name="pdf" type="boolean" truevalue="True" falsevalue="" checked="False" label="Visualising the analysis results" help="output an additional PDF file" />
<param name="binding_matrix" type="boolean" truevalue="True" falsevalue="" checked="False" label="Output binding affinity matrix?" help="Output a table of the binding scores" />
<param name="rdata" type="boolean" truevalue="True" falsevalue="" checked="False" label="Output RData file?" help="Output all the data used by R to construct the plots and tables, can be loaded into R. Default: No"/>
<param name="rscript" type="boolean" truevalue="True" falsevalue="False" checked="False" label="Output Rscript?" help="If this option is set to Yes, the Rscript used will be provided as a text file in the output. Default: No"/>
<param name="analysis_info" type="boolean" truevalue="True" falsevalue="False" checked="False" label="Output analysis info?" help="If this option is set to Yes, information from the dba.count and dba.analyze commmands will be output in a text file. Default: No"/>
</section>
</inputs>
<outputs>
<data name="outfile" format="interval" label="${tool.name} on ${on_string}: Differentially bound sites">
<change_format>
<when input="out.format" value="bed" format="bed" />
<when input="out.format" value="tabular" format="tabular" />
</change_format>
</data>
<data name="plots" format="pdf" label="${tool.name} on ${on_string}: Plots">
<filter>out['pdf']</filter>
</data>
<data name="binding_matrix" format="tabular" from_work_dir="bmatrix.tab" label="${tool.name} on ${on_string}: Binding matrix">
<filter>out['binding_matrix']</filter>
</data>
<data name="rdata" format="rdata" from_work_dir="DiffBind_analysis.RData" label="${tool.name} on ${on_string}: RData file">
<filter>out['rdata']</filter>
</data>
<data name="rscript" format="txt" label="${tool.name} on ${on_string}: Rscript">
<filter>out['rscript']</filter>
</data>
<data name="analysis_info" format="txt" from_work_dir="DiffBind_analysis_info.txt" label="${tool.name} on ${on_string}: Analysis info">
<filter>out['analysis_info']</filter>
</data>
</outputs>
<tests>
<!-- Ensure outputs work -->
<test expect_num_outputs="6">
<repeat name="rep_group">
<param name="groupName" value="Resistant"/>
<param name="peaks" ftype="bed" value="BT474_ER_1.bed.gz,BT474_ER_2.bed.gz"/>
<param name="bamreads" ftype="bam" value="BT474_ER_1.bam,BT474_ER_2.bam" />
</repeat>
<repeat name="rep_group">
<param name="groupName" value="Responsive"/>
<param name="peaks" ftype="bed" value="MCF7_ER_1.bed.gz,MCF7_ER_2.bed.gz"/>
<param name="bamreads" ftype="bam" value="MCF7_ER_1.bam,MCF7_ER_2.bam" />
</repeat>
<param name="scorecol" value="5" />
<param name="format" value="interval"/>
<param name="pdf" value="True" />
<param name="binding_matrix" value="True" />
<param name="rdata" value="True" />
<param name="rscript" value="True"/>
<param name="analysis_info" value="True"/>
<output name="outfile" ftype="interval" value="out_diffbind.interval" />
<output name="plots" value="out_plots.pdf" compare="sim_size" />
<output name="binding_matrix" value="out_binding_matrix.tab" />
<output name="rdata" value="DiffBind_analysis.RData" compare="sim_size"/>
<output name="rscript">
<assert_contents>
<has_text text="write.table"/>
</assert_contents>
</output>
<output name="analysis_info" compare="sim_size" >
<assert_contents>
<has_text text="SessionInfo"/>
</assert_contents>
</output>
</test>
<!-- Ensure control BAMs input works -->
<test expect_num_outputs="1">
<repeat name="rep_group">
<param name="groupName" value="Resistant"/>
<param name="peaks" ftype="bed" value="BT474_ER_1.bed.gz,BT474_ER_2.bed.gz"/>
<param name="bamreads" ftype="bam" value="BT474_ER_1.bam,BT474_ER_2.bam" />
<param name="bamcontrol" ftype="bam" value="input1.bam,input2.bam" />
</repeat>
<repeat name="rep_group">
<param name="groupName" value="Responsive"/>
<param name="peaks" ftype="bed" value="MCF7_ER_1.bed.gz,MCF7_ER_2.bed.gz"/>
<param name="bamreads" ftype="bam" value="MCF7_ER_1.bam,MCF7_ER_2.bam" />
<param name="bamcontrol" ftype="bam" value="input1.bam,input2.bam" />
</repeat>
<param name="scorecol" value="5" />
<param name="format" value="interval"/>
<output name="outfile" ftype="interval" value="out_diffbind_ctrl.interval" />
</test>
<!-- Ensure BED output works -->
<test expect_num_outputs="1">
<repeat name="rep_group">
<param name="groupName" value="Resistant"/>
<param name="peaks" ftype="bed" value="BT474_ER_1.bed.gz,BT474_ER_2.bed.gz"/>
<param name="bamreads" ftype="bam" value="BT474_ER_1.bam,BT474_ER_2.bam" />
</repeat>
<repeat name="rep_group">
<param name="groupName" value="Responsive"/>
<param name="peaks" ftype="bed" value="MCF7_ER_1.bed.gz,MCF7_ER_2.bed.gz"/>
<param name="bamreads" ftype="bam" value="MCF7_ER_1.bam,MCF7_ER_2.bam" />
</repeat>
<param name="scorecol" value="5" />
<param name="format" value="bed"/>
<output name="outfile" ftype="bed" value="out_diffbind.bed" />
</test>
<!-- Ensure tabular output works -->
<test expect_num_outputs="1">
<repeat name="rep_group">
<param name="groupName" value="Resistant"/>
<param name="peaks" ftype="bed" value="BT474_ER_1.bed.gz,BT474_ER_2.bed.gz"/>
<param name="bamreads" ftype="bam" value="BT474_ER_1.bam,BT474_ER_2.bam" />
</repeat>
<repeat name="rep_group">
<param name="groupName" value="Responsive"/>
<param name="peaks" ftype="bed" value="MCF7_ER_1.bed.gz,MCF7_ER_2.bed.gz"/>
<param name="bamreads" ftype="bam" value="MCF7_ER_1.bam,MCF7_ER_2.bam" />
</repeat>
<param name="scorecol" value="5" />
<param name="format" value="tabular"/>
<output name="outfile" ftype="tabular" file="out_diffbind.tab" />
</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 this DiffBind tool requires a minimum of four samples (two groups with two replicates each).**
-----
**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 (BAM files) need to
be associated with each peakset (one for the ChIP data, and optionally another representing
a control sample)
**Groups**
You have to specify the name of the Group and the peak and BAM files for the two Groups you want to compare (e.g Resistant and Responsive) in the tool form above.
Example:
============= =============
**Sample** **Group**
------------- -------------
BT4741 Resistant
BT4742 Resistant
MCF71 Responsive
MCF72 Responsive
============= =============
**Peak files**
Result of your Peak calling experiment in bed format, one file for each sample is required. The peak caller, format and score column can be specified in the tool form above. The default settings expect narrowPeak bed format, which has the score in the 8th column (-log10pvalue), and can be output from MACS2.
Example:
======= ======= ======= =============== ==============
1 2 3 4 **5 (Score)**
======= ======= ======= =============== ==============
chr18 215562 216063 peak_16037 56.11
chr18 311530 312105 peak_16038 222.49
chr18 356656 357315 peak_16039 92.06
chr18 371110 372092 peak_16040 123.86
chr18 395116 396464 peak_16041 1545.39
chr18 399014 400382 peak_16042 1835.19
chr18 499134 500200 peak_16043 748.32
chr18 503518 504552 peak_16044 818.30
chr18 531672 532274 peak_16045 159.30
chr18 568326 569282 peak_16046 601.11
======= ======= ======= =============== ==============
* BAM file which contains the mapped sequencing reads associated with each peakset, one file for each sample is required.
* Optional: Control BAM file representing a control dataset. If used, has to be specified for all samples. Note that the DiffBind authors say control reads are best utilized prior to running DiffBind, at the peak calling stage (e.g. with MACS2) and in blacklists, see this `Bioconductor post`_.
-----
**Outputs**
This tool outputs
* a table of differentially bound sites in Interval, BED or Tabular 0-based format
Optionally, under **Output Options** you can choose to output
* a PDF of plots (Heatmap, PCA, MA, Volcano, Boxplots)
* a binding affinity matrix
* the R script used by this tool
* an RData file of the R objects generated
* a text file with information on the analysis (number of Intervals, FriP scores, method used)
**Differentially Bound Sites**
The default output is Interval format, for information on Interval format see here_. Alternatively, you can choose to output BED or Tabular 0-based format as below. For an explanation of the 0-based and 1-based coordinate systems see this `Biostars post`_.
Example - **Interval format**:
====== ====== ====== ======== ===== ====== ===========================================
Chrom Start End Name Score Strand **Comment**
====== ====== ====== ======== ===== ====== ===========================================
chr18 394599 396513 DiffBind 0 \. 1914|7.15|5.55|7.89|-2.35|7.06e-24|9.84e-21
chr18 111566 112005 DiffBind 0 \. 439|5.71|6.53|3.63|2.89|1.27e-08|8.88e-06
chr18 346463 347342 DiffBind 0 \. 879|5|5.77|3.24|2.52|6.51e-06|0.00303
chr18 399013 400382 DiffBind 0 \. 1369|7.62|7|8.05|-1.04|1.04e-05|0.00364
chr18 371109 372102 DiffBind 0 \. 993|4.63|3.07|5.36|-2.3|8.1e-05|0.0226
====== ====== ====== ======== ===== ====== ===========================================
Columns contain the following data:
* **Chrom**: Chromosome name
* **Start**: Start position of site
* **End**: End position of site
* **Score**: 0
* **Name**: DiffBind
* **Strand**: Strand
* **Comment**: The pipe ("|") separated values in this column correspond to:
* *width*: Length of site
* *Conc*: Mean read concentration over all the samples (the default calculation uses log2 normalized ChIP read counts with control read counts subtracted)
* *Conc_Group1*: Mean concentration over the first group (e.g. Responsive)
* *Conc_Group2*: Mean concentration over second group (e.g. Resistant)
* *Fold*: Fold shows the difference in mean concentrations between the two groups (e.g. Responsive - Resistant), with a positive value indicating increased binding affinity in the first group and a negative value indicating increased binding affinity in the second group.
* *p.value*: P-value confidence measure for identifying these sites as differentially bound
* *FDR*: a multiple testing corrected FDR p-value
Example - **BED format**:
===== ====== ====== ======== ===== ======
Chrom Start End Name Score Strand
===== ====== ====== ======== ===== ======
chr18 394599 396513 DiffBind 0 \.
chr18 111566 112005 DiffBind 0 \.
chr18 346463 347342 DiffBind 0 \.
chr18 399013 400382 DiffBind 0 \.
chr18 371109 372102 DiffBind 0 \.
===== ====== ====== ======== ===== ======
Example - **Tabular format**:
===== ====== ====== ======== ===== ====== ==== =============== ============== ===== ======== ========
Chrom Start End Name Score Strand Conc Conc_Responsive Conc_Resistant Fold p.value FDR
===== ====== ====== ======== ===== ====== ==== =============== ============== ===== ======== ========
chr18 394599 396513 DiffBind 0 \. 7.15 5.55 7.89 -2.35 7.06E-24 9.84E-21
chr18 111566 112005 DiffBind 0 \. 5.71 6.53 3.63 2.89 1.27E-08 8.88E-06
chr18 346463 347342 DiffBind 0 \. 5 5.77 3.24 2.52 6.51E-06 0.00303
chr18 399013 400382 DiffBind 0 \. 7.62 7 8.05 -1.04 1.04E-05 0.00364
chr18 371109 372102 DiffBind 0 \. 4.63 3.07 5.36 -2.3 8.10E-05 0.0226
===== ====== ====== ======== ===== ====== ==== =============== ============== ===== ======== ========
**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. Note that this output is a tabular 0-based format.
Example:
===== ====== ====== ========= ========= ========== ==========
Chrom Start End MCF7_ER_1 MCF7_ER_2 BT474_ER_1 BT474_ER_2
===== ====== ====== ========= ========= ========== ==========
chr18 111567 112005 137.6152 59.87837 29.41393 19.95945
chr18 189223 189652 19.95945 12.60597 11.55547 23.11095
chr18 215232 216063 11.55547 15.75746 31.51493 72.48434
chr18 311530 312172 17.85846 11.55547 54.62588 43.07040
chr18 346464 347342 75.63583 40.96941 21.00995 16.80796
chr18 356560 357362 11.55547 14.70696 57.77737 53.57538
chr18 371110 372102 8.403982 9.454479 81.93882 82.98932
chr18 394600 396513 56.72687 43.07040 510.5419 438.0575
chr18 399014 400382 156.5241 117.6557 558.8648 496.8854
chr18 498906 500200 767.9138 278.3819 196.4430 181.7361
===== ====== ====== ========= ========= ========== ==========
-----
**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 **MACS2**, or using some other criterion (e.g. genomic windows, or all the promoter regions
in a genome). 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
.. _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
.. _`Bioconductor post`: https://support.bioconductor.org/p/69924/
.. _here: https://galaxyproject.org/learn/datatypes/#interval
.. _`Biostars post`: https://www.biostars.org/p/84686/
]]>
</help>
<citations>
<citation type="doi">doi:10.1038/nature10730</citation>
</citations>
</tool>