ChIP-seq Pipeline

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ChIP-Seq pipeline

Here we provide the tools to perform paired end or single read ChIP-Seq analysis including raw data quality control, read mapping, peak calling, differential binding analysis and functional annotation. As input files you may use either zipped fastq-files (.fastq.gz) or mapped read data (.bam files). In case of paired end reads, corresponding fastq files should be named using .R1.fastq.gz and .R2.fastq.gz suffixes.

Pipeline Workflow

All analysis steps are illustrated in the pipeline flowchart . Specify the desired analysis details for your data in the essential.vars.groovy file (see below) and run the pipeline chipseq.pipeline.groovy as described here . A markdown file ChIPreport.Rmd will be generated in the output reports folder after running the pipeline. Subsequently, the ChIPreport.Rmd file can be converted to a final html report using the knitr R-package.

The pipelines includes

  • raw data quality control with FastQC, BamQC and MultiQC
  • mapping reads or read pairs to the reference genome using bowtie2 (default) or bowtie1
  • filter out multimapping reads from bowtie2 output with samtools (optional)
  • identify and remove duplicate reads with Picard MarkDuplicates (optional)
  • generation of bigWig tracks for visualisation of alignment with deeptools bamCoverage. For single end design, reads are extended to the average fragment size
  • characterization of insert size using Picard CollectInsertSizeMetrics (for paired end libraries only)
  • characterize library complexity by PCR Bottleneck Coefficient using the GenomicAlignments R-package (for single read libraries only)
  • characterize phantom peaks by cross correlation analysis using the spp R-package (for single read libraries only)
  • peak calling of IP samples vs. corresponding input controls using MACS2
  • peak annotation using the ChIPseeker R-package (optional)
  • differential binding analysis using the diffbind R-package (optional). For this, input peak files must be given in NGSpipe2go/tools/diffbind/targets_diffbind.txt and contrasts of interest in NGSpipe2go/tools/diffbind/contrasts_diffbind.txt (see below)

Code Snippets

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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/BlackList_Filter/BlackList_Filter.R $BLACKLIST_FILTER_FLAGS;
""","blacklist_filter"
Bpipe From line 24 of ChIPseq/blacklist_filter.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    bowtie $BOWTIE_FLAGS ${bowtie1_vars.ref} $BOWTIE_INPUT | samtools view $SAMTOOLS_VIEW_FLAGS - | samtools sort $SAMTOOLS_SORT_FLAGS -T \${TMP}/\$(basename $output.prefix)_bowtie1_sort - > $output
""","bowtie1"
Bpipe SAMtools Bowtie 2 From line 37 of ChIPseq/bowtie1.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    bowtie2 $BOWTIE2_FLAGS $BOWTIE2_INPUT | samtools view $SAMTOOLS_VIEW_FLAGS - | samtools sort $SAMTOOLS_SORT_FLAGS -T \${TMP}/\$(basename $output.prefix) - > $output;
""","bowtie2"
Bpipe SAMtools Bowtie 2 From line 37 of ChIPseq/bowtie2.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/diffbind/diffbind2.R $DIFFBIND_FLAGS
""","diffbind2"
Bpipe From line 41 of ChIPseq/diffbind2.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/diffbind/diffbind3.R $DIFFBIND_FLAGS
""","diffbind3"
Bpipe From line 47 of ChIPseq/diffbind3.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    samtools view $FILBOWTIE2_FLAGS -bhu ${input} | samtools sort -@ $filbowtie2unique_vars.samtools_threads -T \${TMP}/\$(basename $output.prefix) -o ${input.prefix}_mmremoved.bam - &&
    if [[ "${dupremoval_vars.remove_pcr_dups}" == "true" ]]; then
        echo "Removing PCR duplicates";
        bam dedup --in ${input.prefix}_mmremoved.bam --out ${output} --log ${LOGS}/filbowtie2unique/\$(basename ${output.prefix})_dupmetrics.log --noPhoneHome --force --rmDups;
        rm ${input.prefix}_mmremoved.bam;
    else
        echo "Not removing the PCR duplicates";
        mv ${input.prefix}_mmremoved.bam ${output};
    fi;

""","filbowtie2unique"
Bpipe SAMtools From line 19 of ChIPseq/filbowtie2unique.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/GO_Enrichment/GREAT.R $GREAT_FLAGS
""","GREAT"
Bpipe From line 26 of ChIPseq/GREAT.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    touch $output;
    if [ ! -e ${ipstrength_vars.targets} ]; then
        echo "Targets file ${ipstrength_vars.targets} doesn't exist" >> $output &&
        exit 0;
    fi;

    BAM=\$(basename $input) &&           
    extension="\${BAM#*.}" &&
    BAM="\${BAM%%.*}" &&
    grep "^\$BAM" ${ipstrength_vars.targets} | while read -r TARGET; do
        IP=\$(       echo $TARGET | tr '\t' ' ' | cut -f1 -d" ").\$extension &&
        IPname=\$(   echo $TARGET | tr '\t' ' ' | cut -f2 -d" ") &&
        INPUT=\$(    echo $TARGET | tr '\t' ' ' | cut -f3 -d" ").\$extension &&
        INPUTname=\$(echo $TARGET | tr '\t' ' ' | cut -f4 -d" ");

        if [ "\$BAM" != "\$INPUT" ]; then
            if [ "\$INPUT" != "none.\$extension" ]; then
              echo "\${IPname} vs \${INPUTname}" >> $output ;
              Rscript ${PIPELINE_ROOT}/tools/ENCODEqc/IPstrength.R ${ipstrength_vars.mapped}/\$IP \$IPname ${ipstrength_vars.mapped}/\$INPUT \$INPUTname $subdir\${IPname}.vs.\${INPUTname}_ipstrength ${ipstrength_vars.bsgenome};
              if [ \$? -ne 0 ]; then rm $output; fi;
              find . -maxdepth 1 -name "$subdir\${IPname}.vs.\${INPUTname}_ipstrength*" -exec sh -c 'mv "\$1" "$output.dir/\${1#./$subdir}"' _ {} \\;;
            else
              echo "\${IPname} skipped because no input" >> $output ;
          fi;
        fi;
    done
""","ipstrength"
Bpipe From line 15 of ChIPseq/ipstrength.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    touch $output;
    if [ ! -e ${macs2_vars.targets} ]; then
        echo "Targets file ${macs2_vars.targets} doesn't exist" >> $output &&
        exit 0;
    fi;

    BAM=\$(basename $input) &&           
    extension="\${BAM#*.}" &&
    BAM="\${BAM%%.*}" &&
    grep "^\$BAM" ${macs2_vars.targets} | while read -r TARGET; do
        IP=\$(       echo $TARGET | tr '\t' ' ' | cut -f1 -d" ").\$extension &&
        IPname=\$(   echo $TARGET | tr '\t' ' ' | cut -f2 -d" ") &&
        INPUT=\$(    echo $TARGET | tr '\t' ' ' | cut -f3 -d" ").\$extension &&
        INPUTname=\$(echo $TARGET | tr '\t' ' ' | cut -f4 -d" ");

        if [ "\$BAM" != "\$INPUT" ]; then
            if [ "\$INPUT" != "none.\$extension" ]; then
                NAMEoutput="\${IPname}.vs.\${INPUTname}" &&
                INPUTflag="-c ${macs2_vars.mapped}/\$INPUT";
            else
                NAMEoutput="\${IPname}" &&
                INPUTflag="";
            fi &&   
            echo "\$NAMEoutput" >> $output &&
            macs2 callpeak -t ${macs2_vars.mapped}/\$IP \$INPUTflag -n $subdir\${NAMEoutput}_macs2 $MACS2_FLAGS ;
            if [ \$? -ne 0 ]; then rm $output; fi ;
            find . -maxdepth 1 -name "$subdir\${NAMEoutput}_macs2*" -exec sh -c 'mv "\$1" "$output.dir/\${1#./$subdir}"' _ {} \\;;
        fi;
    done
""","macs2"
Bpipe macs2 From line 22 of ChIPseq/macs2.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/BlackList_Filter/make_greylist.R $MAKE_GREYLIST_FLAGS;
""","make_greylist"
Bpipe From line 24 of ChIPseq/make_greylist.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/ENCODEqc/PBC.R $input && mv ${input.prefix}_PBC.csv $output.dir
""","pbc"
Bpipe From line 15 of ChIPseq/pbc.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/Peak_Annotation/Peak_Annotation.R $PEAK_ANNOTATION_FLAGS;
""","peak_annotation"
Bpipe From line 26 of ChIPseq/peak_annotation.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/ENCODEqc/phantompeak.R $input \$(basename $input.prefix) $PHANTOMPEAK_FLAGS &&

    mv \$(basename $input.prefix)_phantompeak.* $output.dir
""","phantompeak"
Bpipe From line 23 of ChIPseq/phantompeak.groovy 
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       exec """
           ${PREAMBLE} &&

           cp ${PIPELINE_ROOT}/tools/reports/shiny_chipseq_reporting_tool/ChIP.shinyrep.helpers.R ${REPORTS} &&
           cp ${PIPELINE_ROOT}/tools/reports/shiny_chipseq_reporting_tool/styles.css ${REPORTS}              &&

           if [ -e "${REPORTS}/ChIPreport.Rmd" ]; then
               echo 'ChIPreport.Rmd already exists. Older copy will be kept and not overwritten';
           else
               cp ${PIPELINE_ROOT}/tools/reports/shiny_chipseq_reporting_tool/ChIPreport.Rmd ${REPORTS};
           fi &&

           PROJECT=\$(basename ${shinyReports_vars.project})                              &&
           sed -i "2,2s/SHINYREPS_PROJECT/\${PROJECT}/" ${REPORTS}/ChIPreport.Rmd &&

           echo "SHINYREPS_PROJECT=${shinyReports_vars.project}" >  $output &&
           echo "SHINYREPS_LOG=${shinyReports_vars.log}"         >> $output &&
           echo "SHINYREPS_QC=${shinyReports_vars.qc}"           >> $output &&
           echo "SHINYREPS_RES=${shinyReports_vars.res}"         >> $output &&
           echo "SHINYREPS_TARGET=${shinyReports_vars.target}"   >> $output &&
           echo "SHINYREPS_PAIRED=${shinyReports_vars.paired}"      >> $output &&
           echo "SHINYREPS_RUN_CUTADAPT=${shinyReports_vars.run_cutadapt}"  >> $output &&
           echo "SHINYREPS_RUN_PEAK_ANNOTATION=${shinyReports_vars.run_peakanno}"  >> $output &&
           echo "SHINYREPS_RUN_DIFFBIND=${shinyReports_vars.run_diffbind}"  >> $output &&
           echo "SHINYREPS_RUN_ENRICHMENT=${shinyReports_vars.run_enrich}"  >> $output &&
	    echo "SHINYREPS_CUTADAPT_STATS=${shinyReports_vars.cutadapt_stats}" >> $output &&
	    echo "SHINYREPS_SAMTOOLSCOV_OUT=${shinyReports_vars.samtoolscov_out}" >> $output &&
	    echo "SHINYREPS_BOWTIE_LOG=${shinyReports_vars.bowtie_log}"      >> $output &&
           echo "SHINYREPS_BAMINDEX_LOG=${shinyReports_vars.bamindex_log}"  >> $output &&
           echo "SHINYREPS_MARKDUPS_LOG=${shinyReports_vars.markdups_log}"  >> $output &&
           echo "SHINYREPS_EXTEND_LOG=${shinyReports_vars.extend_log}"      >> $output &&
           echo "SHINYREPS_FASTQC=${shinyReports_vars.fastqc}"   >> $output &&
           echo "SHINYREPS_FASTQC_LOG=${shinyReports_vars.fastqc_log}"   >> $output &&
           echo "SHINYREPS_FASTQC_SUMMARIZED=${shinyReports_vars.fastqc_summarized}" >> $output &&
           echo "SHINYREPS_FASTQSCREEN_OUT=${shinyReports_vars.fastqscreen_out}" >> $output &&
           echo "SHINYREPS_FASTQSCREEN_LOG=${shinyReports_vars.fastqscreen_log}" >> $output &&
           echo "SHINYREPS_FASTQSCREEN_PERC=${shinyReports_vars.fastqscreen_perc}" >> $output &&
           echo "SHINYREPS_INSERTSIZE=${shinyReports_vars.insertsize}" >> $output &&
           echo "SHINYREPS_IPSTRENGTH=${shinyReports_vars.ipstrength}"      >> $output &&
           echo "SHINYREPS_IPSTRENGTH_LOG=${shinyReports_vars.ipstrength_log}"      >> $output &&
           echo "SHINYREPS_PBC=${shinyReports_vars.pbc}"         >> $output &&
           echo "SHINYREPS_PHANTOMPEAK=${shinyReports_vars.phantompeak}"    >> $output &&
           echo "SHINYREPS_PHANTOM_LOG=${shinyReports_vars.phantom_log}"    >> $output &&
           echo "SHINYREPS_MACS2=${shinyReports_vars.macs2}"     >> $output &&
           echo "SHINYREPS_MACS2_LOG=${shinyReports_vars.macs2_log}"         >> $output &&
           echo "SHINYREPS_BLACKLIST_FILTER=${shinyReports_vars.blacklist_filter}" >> $output &&
           echo "SHINYREPS_UPSETPLOT=${shinyReports_vars.upset}" >> $output &&
           echo "SHINYREPS_PREFIX=${shinyReports_vars.prefix}"   >> $output &&
           echo "SHINYREPS_PLOTS_COLUMN=${shinyReports_vars.plots_column}" >> $output &&
           echo "SHINYREPS_PEAK_ANNOTATION=${shinyReports_vars.peak_annotation}" >> $output &&
           echo "SHINYREPS_DB=${shinyReports_vars.db}"   >> $output &&
           echo "SHINYREPS_GREAT=${shinyReports_vars.great}" >> $output &&
           echo "SHINYREPS_DIFFBIND=${shinyReports_vars.diffbind}" >> $output           &&
           echo "SHINYREPS_TRACKHUB_DONE=${shinyReports_vars.trackhub_done}" >> $output &&
           echo "SHINYREPS_TOOL_VERSIONS=${shinyReports_vars.tool_versions}" >> $output
       ""","shinyReports"
Bpipe From line 13 of ChIPseq/shinyreports.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/upsetPlot/upsetPlot.R $UPSET_FLAGS;
""","upsetPlot"
Bpipe From line 25 of ChIPseq/upsetPlot.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    bamCoverage $BAMCOVERAGE_FLAGS --bam $input -o ${output};
""","bamCoverage"
Bpipe DeepTools From line 20 of NGS/bamcoverage.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    samtools index $input
""","BAMindexer"
Bpipe SAMtools From line 15 of NGS/bamindexer.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    bamqc $BAMQC_FLAGS -o $output.dir $input
""","BamQC"
Bpipe From line 16 of NGS/bamqc.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    cutadapt $CUTADAPT_FLAGS $CUTADAPT_FLAGS_PAIRED $CUTADAPT_FLAGS_TOOLONG --too-short-output=\${TMP}/${SAMPLENAME_BASE}.cutadapt_discarded.fastq.gz --output=\${TMP}/${SAMPLENAME_BASE}.cutadapt.fastq.gz $input1 $CUTADAPT_INPUT_SECOND 1> ${CUTADAPT_STATSDIR}/${SAMPLENAME_BASE_PRUNED}.cutadapt.log &&

    mv -t ${CUTADAPT_DISCARDED_DIR}/ \${TMP}/${SAMPLENAME_BASE_PRUNED}*cutadapt_discarded*.fastq.gz &&
    mv -t $output.dir \${TMP}/${SAMPLENAME_BASE_PRUNED}*cutadapt.fastq.gz
""","Cutadapt"
Bpipe Cutadapt From line 50 of NGS/cutadapt.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    CHRSIZES="\${TMP}/\$(basename ${input.prefix}).extend.chrsizes"  &&
    samtools idxstats ${input} | cut -f1-2 > "\${CHRSIZES}" &&
    bedtools bamtobed -split -i $input | bedtools slop -g "\${CHRSIZES}" -l 0 -r ${extend_vars.fraglen} -s | bedtools bedtobam -ubam -g "\${CHRSIZES}" | samtools sort $SAMTOOLS_SORT_FLAGS -T \${TMP}/\$(basename $output.prefix) - > $output &&
    samtools index $output
""","extend"
Bpipe SAMtools BEDTools From line 17 of NGS/extend.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    fastqc --extract $FASTQC_FLAGS -o $output.dir $inputs
""","FastQC"
Bpipe FastQC From line 18 of NGS/fastqc.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    if [ ! -e "$output.prefix" ]; then
        mkdir $output.prefix;
    fi &&
    fastqreference=${FastqScreen_vars.conf};
    references=(\${fastqreference//,/ });
    for i in "\${!references[@]}"; do
        reference=(\${references[i]//::/ });
        echo -e "DATABASE\t\${reference[0]}\t\${reference[1]}" >> $output.prefix/fastqscreen.conf;
    done;
    fastq_screen $FASTQSCREEN_FLAGS --conf $output.prefix/fastqscreen.conf --outdir $output.prefix $inputs;
    touch $outputs
""","FastqScreen"
Bpipe From line 18 of NGS/fastqscreen.groovy 
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exec """
     ${TOOL_ENV} &&
     ${PREAMBLE} &&

    chroms=\$(cut -f1 ${FilterChr_vars.file}) && 
    samtools view -@ ${FilterChr_vars.threads} -b $input \$chroms > $output &&
    samtools index $output
""","FilterChr"
Bpipe SAMtools From line 14 of NGS/filterchromosomes.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    java ${InsertSize_vars.java_flags} -jar \${PICARD} CollectInsertSizeMetrics $INSERTSIZE_FLAGS INPUT=$input OUTPUT=$output HISTOGRAM_FILE=${output.prefix}_hist.pdf
""","InsertSize"
Bpipe From line 20 of NGS/insertsize.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    java ${MarkDups_vars.java_flags} -jar \${PICARD} MarkDuplicates $MARKDUPS_FLAGS INPUT=$input OUTPUT=$output METRICS_FILE=${input.prefix}_dupmarked_dupmetrics.tsv
""","MarkDups"
Bpipe From line 19 of NGS/markdups.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    multiqc $ESSENTIAL_PROJECT $MultiQC_FLAGS -o $output.dir
""","MULTIQC"
Bpipe MultiQC From line 16 of NGS/multiqc.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    java ${RmDups_vars.java_flags} -jar \${PICARD} MarkDuplicates $RMDUPS_FLAGS INPUT=$input OUTPUT=$output METRICS_FILE=${input.prefix}_duprm_dupmetrics.tsv TMP_DIR=\${TMP}
""","RmDups"
Bpipe From line 19 of NGS/rmdups.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    samtools coverage $SAMTOOLSCOV_FLAGS -o $output $input
""","samtoolscov"
Bpipe SAMtools From line 16 of NGS/samtoolscov.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/trackhub/Configure_Trackhub.R $TRACKHUB_FLAGS
""","trackhub_config"
Bpipe From line 26 of NGS/trackhub_config.groovy 
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exec """
    ${TOOL_ENV} &&
    ${PREAMBLE} &&

    Rscript ${PIPELINE_ROOT}/tools/trackhub/Make_Trackhub.R $TRACKHUB_FLAGS
""","trackhub"
Bpipe From line 17 of NGS/trackhub.groovy
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://gitlab.rlp.net/imbforge/NGSpipe2go/-/tree/master/pipelines/ChIPseq
Name: chip-seq
Version: Version 1
Badge:
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Other Versions:
Version 2
Downloaded: 0
Copyright: Public Domain
License: GNU General Public License v3.0
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