Analysis of Chromosome Conformation Capture data (Hi-C)

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Introduction

nf-core/hic is a bioinformatics best-practice analysis pipeline for Analysis of Chromosome Conformation Capture data (Hi-C).

The pipeline is built using Nextflow , a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It uses Docker/Singularity containers making installation trivial and results highly reproducible. The Nextflow DSL2 implementation of this pipeline uses one container per process which makes it much easier to maintain and update software dependencies. Where possible, these processes have been submitted to and installed from nf-core/modules in order to make them available to all nf-core pipelines, and to everyone within the Nextflow community!

On release, automated continuous integration tests run the pipeline on a full-sized dataset on the AWS cloud infrastructure. This ensures that the pipeline runs on AWS, has sensible resource allocation defaults set to run on real-world datasets, and permits the persistent storage of results to benchmark between pipeline releases and other analysis sources.The results obtained from the full-sized test can be viewed on the nf-core website .

Pipeline summary

  1. Read QC ( FastQC )

  2. Hi-C data processing

    1. HiC-Pro

      1. Mapping using a two steps strategy to rescue reads spanning the ligation sites ( bowtie2 )

      2. Detection of valid interaction products

      3. Duplicates removal

      4. Generate raw and normalized contact maps ( iced )

  3. Create genome-wide contact maps at various resolutions ( cooler )

  4. Contact maps normalization using balancing algorithm ( cooler )

  5. Export to various contact maps formats ( HiC-Pro , cooler )

  6. Quality controls ( HiC-Pro , HiCExplorer )

  7. Compartments calling ( cooltools )

  8. TADs calling ( HiCExplorer , cooltools )

  9. Quality control report ( MultiQC )

Usage

Note If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data.

First, prepare a samplesheet with your input data that looks as follows:

samplesheet.csv : 

sample,fastq_1,fastq_2
HIC_ES_4,SRR5339783_1.fastq.gz,SRR5339783_2.fastq.gz

Each row represents a pair of fastq files (paired end). Now, you can run the pipeline using: 

nextflow run nf-core/hic \
 -profile <docker/singularity/.../institute> \
 --input samplesheet.csv \
 --genome GRCh37 \
 --outdir <OUTDIR>

Warning: Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters ; see docs .

For more details, please refer to the usage documentation and the parameter documentation .

Pipeline output

To see the the results of a test run with a full size dataset refer to the results tab on the nf-core website pipeline page. For more details about the output files and reports, please refer to the output documentation .

Credits

nf-core/hic was originally written by Nicolas Servant.

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines .

For further information or help, don't hesitate to get in touch on the Slack #hic channel (you can join with this invite ).

Citations

If you use nf-core/hic for your analysis, please cite it using the following doi: doi: 10.5281/zenodo.2669512

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x .

Code Snippets

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"""
cooltools genome binnify --all-names ${chrsize} ${resolution} > genome_bins.txt
cooltools genome gc genome_bins.txt ${fasta} > genome_gc.txt
cooltools eigs-cis ${args} -o ${prefix}_compartments ${cool}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    cooltools: \$(cooltools --version 2>&1 | grep version | sed 's/cooltools, version //')
END_VERSIONS
"""
NextFlow cooltools From line 26 of cooltools/eigscis.nf 
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"""
cooltools insulation ${cool} ${args} > ${prefix}_insulation.tsv

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    cooltools: \$(cooltools --version 2>&1 | sed 's/cooltools, version //')
END_VERSIONS
"""
NextFlow cooltools From line 24 of cooltools/insulation.nf 
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"""
hicFindTADs --matrix ${cool} \
    --outPrefix ${prefix}_hicfindtads \
    ${args} \
    --numberOfProcessors ${task.cpus}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    hicexplorer: \$(hicFindTADs --version 2>&1 | sed 's/hicFindTADs //')
END_VERSIONS
"""
NextFlow HiCExplorer From line 23 of hicexplorer/hicFindTADs.nf 
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"""
hicPlotDistVsCounts --matrices ${cool} \
                    --plotFile ${prefix}_distcount.png \
                    --outFileData ${prefix}_distcount.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    hicexplorer: \$(hicPlotDistVsCounts --version 2>&1 | sed 's/hicPlotDistVsCounts //')
END_VERSIONS
"""
NextFlow HiCExplorer From line 24 of hicexplorer/hicPlotDistVsCounts.nf 
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"""
samtools merge -@ ${task.cpus} \\
    -f ${prefix}_bwt2merged.bam \\
    ${bam1} ${bam2}

samtools sort -@ ${task.cpus} -m 800M \\
    -n \\
    -o ${prefix}_bwt2merged.sorted.bam \\
    ${prefix}_bwt2merged.bam

mv ${prefix}_bwt2merged.sorted.bam ${prefix}_bwt2merged.bam

echo "## ${prefix}" > ${prefix}.mapstat
echo -n "total_${tag}\t" >> ${prefix}.mapstat
samtools view -c ${prefix}_bwt2merged.bam >> ${prefix}.mapstat
echo -n "mapped_${tag}\t" >> ${prefix}.mapstat
samtools view -c -F 4 ${prefix}_bwt2merged.bam >> ${prefix}.mapstat
echo -n "global_${tag}\t" >> ${prefix}.mapstat
samtools view -c -F 4 ${bam1} >> ${prefix}.mapstat
echo -n "local_${tag}\t"  >> ${prefix}.mapstat
samtools view -c -F 4 ${bam2} >> ${prefix}.mapstat

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""
NextFlow SAMtools From line 21 of hicpro/bowtie2_merge.nf 
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"""
build_matrix \\
    --matrix-format upper  \\
    --binsize ${resolution} \\
    --chrsizes ${chrsize} \\
    --ifile ${vpairs} \\
    --oprefix ${prefix}
"""
NextFlow From line 19 of hicpro/build_contact_maps.nf 
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"""
mergeSAM.py -f ${bam[0]} -r ${bam[1]} -o ${prefix}_bwt2pairs.bam ${args}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(echo \$(python --version 2>&1) | sed 's/Python //')
END_VERSIONS
"""
NextFlow From line 21 of hicpro/combine_mates.nf 
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"""
echo "## ${prefix}" > ${prefix}.mapstat
echo -n "total_${tag}\t" >> ${prefix}.mapstat
samtools view -c ${bam} >> ${prefix}.mapstat
echo -n "mapped_${tag}\t" >> ${prefix}.mapstat
samtools view -c -F 4 ${bam} >> ${prefix}.mapstat
echo -n "global_${tag}\t" >> ${prefix}.mapstat
samtools view -c -F 4 ${bam} >> ${prefix}.mapstat
echo -n "local_${tag}\t0"  >> ${prefix}.mapstat
"""
NextFlow SAMtools From line 21 of hicpro/dnase_mapping_stats.nf 
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"""
digest_genome.py -r ${res_site} -o restriction_fragments.bed ${fasta}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(echo \$(python --version 2>&1) | sed 's/Python //')
END_VERSIONS
"""
NextFlow From line 19 of hicpro/get_restriction_fragments.nf 
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"""
mapped_2hic_dnase.py \\
    -r ${bam} \\
    ${args}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(echo \$(python --version 2>&1) | sed 's/Python //')
END_VERSIONS
"""
NextFlow From line 20 of hicpro/get_valid_interaction_dnase.nf 
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"""
mapped_2hic_fragments.py \\
    -f ${resfrag} \\
    -r ${bam} \\
    --all \\
    ${args}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(echo \$(python --version 2>&1) | sed 's/Python //')
END_VERSIONS
"""
NextFlow From line 25 of hicpro/get_valid_interaction.nf 
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"""
##columns: readID chr1 pos1 chr2 pos2 strand1 strand2
awk '{OFS="\t";print \$1,\$2,\$3,\$5,\$6,\$4,\$7}' $vpairs | bgzip -c > ${prefix}_contacts.pairs.gz
##sort -k2,2 -k4,4 -k3,3n -k5,5n ${prefix}_contacts.pairs | bgzip -c > ${prefix}_contacts.pairs.gz
pairix -f ${prefix}_contacts.pairs.gz

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    pairix: \$(echo \$(pairix 2>&1 | grep Version | sed -e 's/Version: //'))
END_VERSIONS
"""
NextFlow Pairix From line 20 of hicpro/hicpro2pairs.nf 
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"""
mkdir -p ${meta.id}
merge_statfiles.py -f ${fstat} > ${meta.id}.${ext}
cp *${ext} ${meta.id}/

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(echo \$(python --version 2>&1) | sed 's/Python //')
END_VERSIONS
"""
NextFlow From line 22 of hicpro/merge_stats.nf 
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"""
hicpro_merge_validpairs.sh ${args} -p ${prefix} ${vpairs}

## For MultiQC
mkdir -p ${prefix}
cp ${prefix}_allValidPairs.mergestat ${prefix}/

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    sort: \$(echo \$(sort --version 2>&1 | head -1 | awk '{print \$NF}' 2>&1))
END_VERSIONS
"""
NextFlow From line 22 of hicpro/merge_valid_interaction.nf 
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"""
ice --filter_low_counts_perc ${params.ice_filter_low_count_perc} \
    --results_filename ${prefix}_iced.matrix \
    --filter_high_counts_perc ${params.ice_filter_high_count_perc} \
    --max_iter ${params.ice_max_iter} --eps ${params.ice_eps} --remove-all-zeros-loci --output-bias 1 --verbose 1 ${rmaps}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(echo \$(python --version 2>&1) | sed 's/Python //')
    iced: \$(python -c "import iced; print(iced.__version__)")
END_VERSIONS
"""
NextFlow From line 20 of hicpro/run_ice.nf 
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"""
zcat ${reads} > tmp.fastq
cutsite_trimming --fastq tmp.fastq \\
    --cutsite ${motif[0]} \\
    --out ${reads.simpleName}_trimmed.fastq
gzip ${reads.simpleName}_trimmed.fastq
/bin/rm -f tmp.fastq

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    gzip: \$(echo \$(gzip --version 2>&1) | head -1 | cut -d" " -f2)
END_VERSIONS
"""
NextFlow From line 19 of hicpro/trim_reads.nf 
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"""
multiqc -f $args .

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    multiqc: \$( multiqc --version | sed -e "s/multiqc, version //g" )
END_VERSIONS
"""
NextFlow MultiQC From line 27 of local/multiqc.nf 
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"""
check_samplesheet.py \\
    $samplesheet \\
    samplesheet.valid.csv

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(python --version | sed 's/Python //g')
END_VERSIONS
"""
NextFlow From line 21 of local/samplesheet_check.nf 
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"""
cat ${bedpe} | awk '{OFS="\t"; print \$1,\$2,\$3}' > ${prefix}_raw.txt
cat ${bedpe} | awk '{OFS="\t"; print \$1,\$2,\$4}' > ${prefix}_balanced.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    cooler: \$(awk --version | head -1 | cut -f1 -d, | sed -e 's/GNU Awk //')
END_VERSIONS
"""
NextFlow From line 23 of local/split_cooler_dump.nf 
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"""
INDEX=`find -L ./ -name "*.rev.1.bt2" | sed "s/\\.rev.1.bt2\$//"`
[ -z "\$INDEX" ] && INDEX=`find -L ./ -name "*.rev.1.bt2l" | sed "s/\\.rev.1.bt2l\$//"`
[ -z "\$INDEX" ] && echo "Bowtie2 index files not found" 1>&2 && exit 1

bowtie2 \\
    -x \$INDEX \\
    $reads_args \\
    --threads $task.cpus \\
    $unaligned \\
    $args \\
    2> ${prefix}.bowtie2.log \\
    | samtools $samtools_command $args2 --threads $task.cpus -o ${prefix}.bam -

if [ -f ${prefix}.unmapped.fastq.1.gz ]; then
    mv ${prefix}.unmapped.fastq.1.gz ${prefix}.unmapped_1.fastq.gz
fi

if [ -f ${prefix}.unmapped.fastq.2.gz ]; then
    mv ${prefix}.unmapped.fastq.2.gz ${prefix}.unmapped_2.fastq.gz
fi

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bowtie2: \$(echo \$(bowtie2 --version 2>&1) | sed 's/^.*bowtie2-align-s version //; s/ .*\$//')
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
    pigz: \$( pigz --version 2>&1 | sed 's/pigz //g' )
END_VERSIONS
"""
NextFlow SAMtools Bowtie 2 From line 42 of align/main.nf 
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"""
mkdir bowtie2
bowtie2-build $args --threads $task.cpus $fasta bowtie2/${fasta.baseName}
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bowtie2: \$(echo \$(bowtie2 --version 2>&1) | sed 's/^.*bowtie2-align-s version //; s/ .*\$//')
END_VERSIONS
"""
NextFlow Bowtie 2 From line 22 of build/main.nf 
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"""
cp ${cool} ${prefix}.${extension}

cooler balance \\
    $args \\
    -p ${task.cpus} \\
    ${prefix}.${extension}${suffix}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    cooler: \$(cooler --version 2>&1 | sed 's/cooler, version //')
END_VERSIONS
"""
NextFlow cooler From line 26 of balance/main.nf 
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"""
cooler cload \\
    $args \\
    $nproc \\
    ${chromsizes}:${cool_bin} \\
    $pairs \\
    ${prefix}.cool

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    cooler: \$(cooler --version 2>&1 | sed 's/cooler, version //')
END_VERSIONS
"""
NextFlow cooler From line 26 of cload/main.nf 
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"""
cooler dump \\
    $args \\
    -o ${prefix}.bedpe \\
    $cool$suffix

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    cooler: \$(cooler --version 2>&1 | sed 's/cooler, version //')
END_VERSIONS
"""
NextFlow cooler From line 24 of dump/main.nf 
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"""
cooler makebins \\
    $args \\
    ${chromsizes} \\
    ${cool_bin} > ${prefix}.bed

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    cooler: \$(cooler --version 2>&1 | sed 's/cooler, version //')
END_VERSIONS
"""
NextFlow cooler From line 23 of makebins/main.nf 
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"""
cooler zoomify \\
    $args \\
    -n $task.cpus \\
    -o ${prefix}.mcool \\
    $cool

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    cooler: \$(cooler --version 2>&1 | sed 's/cooler, version //')
END_VERSIONS
"""
NextFlow cooler From line 23 of zoomify/main.nf 
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"""
samtools faidx $fasta
cut -f 1,2 ${fasta}.fai > ${fasta}.sizes

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    getchromsizes: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""
NextFlow SAMtools From line 24 of getchromsizes/main.nf 
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"""
touch ${fasta}.fai
touch ${fasta}.sizes

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    getchromsizes: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""
NextFlow From line 35 of getchromsizes/main.nf 
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"""
printf "%s %s\\n" $rename_to | while read old_name new_name; do
    [ -f "\${new_name}" ] || ln -s \$old_name \$new_name
done
fastqc $args --threads $task.cpus $renamed_files

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fastqc: \$( fastqc --version | sed -e "s/FastQC v//g" )
END_VERSIONS
"""
NextFlow FastQC From line 28 of fastqc/main.nf 
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"""
touch ${prefix}.html
touch ${prefix}.zip

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fastqc: \$( fastqc --version | sed -e "s/FastQC v//g" )
END_VERSIONS
"""
NextFlow FastQC From line 42 of fastqc/main.nf
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://nf-co.re/hic
Name: hic
Version: 2.1.0
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