Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag

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bcd_nano_cutntag
bcd_nano_cutntag
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Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag

Marek Bartosovic, Goncalo Castelo-Branco

Code repository related to preprint.

Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag Marek Bartosovic, Gonçalo Castelo-Branco bioRxiv 2022.03.08.483459; doi: https://doi.org/10.1101/2022.03.08.483459

Data availability

Processed files - Seurat objects, fragments file (cellranger), bigwig tracks per cluster and .h5 matrices are available as supplementary files in the GEO repository https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE198467

Reproducing the analysis

Step 1: prepare environment

conda environment is provided in env/environment.yaml 

conda env create -f env/environment.yaml

Additional package dependencies that need to be installed: 

R
install.packages(c('argparse','ggplot2','funr','Signac','scales','Seurat','rmarkdown','mclust','GGally','BiocManager','patchwork','markdown','UpSetR','pheatmap','viridis','purrr','Rmagic','devtools','raster'))
BiocManager::install(c('ensembldb','EnsDb.Mmusculus.v79','GenomeInfoDb', 'GenomicRanges', 'IRanges', 'Rsamtools','BiocGenerics','rtracklayer','limma','slingshot','BiocGenerics', 'DelayedArray', 'DelayedMatrixStats','limma', 'S4Vectors', 'SingleCellExperiment','SummarizedExperiment', 'batchelor', 'Matrix.utils')))

Have seqtk installed in $PATH

https://github.com/lh3/seqtk

Install papermill for cli for jupyter notebooks

python3 -m pip install papermill

Step 2: Download the data

use fasterq-dump or alternative to download the fastq files

https://github.com/ncbi/sra-tools/wiki/HowTo:-fasterq-dump

GEO repository

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE198467

Step 3: Clone the github repo with analysis code 

git clone https://github.com/mardzix/bcd_nano_CUTnTag

Step 4: Modify config

Change config/config.yaml and specify path to

  1. Absolute path to fastq files

  2. Specific path to the tmp folder (Create any folder, e.g. in home)

  3. Specify conda environment to use

  4. Modify path to cellranger reference

Step 5: Run the pipeline

Pipeline is implemented in workflow management software Snakemake

Change the cluster-specific profile to your preference (e.g. slurm, condor etc.), or run without profile

For some example profiles see:

  • https://snakemake.readthedocs.io/en/stable/executing/cli.html#profiles

  • https://github.com/Snakemake-Profiles/slurm

  • https://github.com/Snakemake-Profiles/htcondor 

snakemake --snakefile code/workflow/Snakefile_single_modality.smk --cores 16 --profile htcondor -p

Johannes Köster, Sven Rahmann, Snakemake—a scalable bioinformatics workflow engine, Bioinformatics, Volume 28, Issue 19, 1 October 2012, Pages 2520–2522, https://doi.org/10.1093/bioinformatics/bts480

Code Snippets

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shell:
    'Rscript {input.script} -i {input.seurat} -m {wildcards.combination} -o {output}'
SnakeMake From line 25 of workflow/Snakefile_multimodal.smk 
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shell:
    "Rscript -e \"rmarkdown::render(input='{input.notebook}', "
    "                                output_file = '{params.report}', "
    "                                params=list(seurat = '{params.seurat_in}', "
    "                                           output = '{params.seurat_out}'))\" "
SnakeMake From line 39 of workflow/Snakefile_multimodal.smk 
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shell:
    "Rscript {input.script} --seurat {input.seurat} --clusters OPC MOL --reduction wnn.umap --out {output.seurat}"
SnakeMake From line 52 of workflow/Snakefile_multimodal.smk 
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shell:
    'Rscript {input.script} -i {input.seurat} -o {output.seurat} {params.clusters} -d {params.idents} -m {params.modalities} -a {params.assay} -t {input.seurat_pt}'
SnakeMake From line 68 of workflow/Snakefile_multimodal.smk 
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shell:
    "fasterq-dump -t {params.tmp} -f -e {threads} --split-files --include-technical -o {params.out} {wildcards.SRA}"
SnakeMake From line 39 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    "gzip {input}"
SnakeMake From line 47 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    "mv {input} {output}"
SnakeMake From line 55 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'rm -r results/nbiotech_data/cellranger/{wildcards.sample}/; '
    'cd results/nbiotech_data/cellranger/; '
    '/data/bin/cellranger-atac count --id {wildcards.sample} --sample {params.sample} --reference {params.cellranger_ref} --fastqs {params.fastq_dir}'
SnakeMake From line 70 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'bamCoverage -b {input.cellranger_bam} -o {output.bigwig} -p {threads} --minMappingQuality 5 '
    ' --binSize 50 --centerReads --smoothLength 250 --normalizeUsing RPKM --ignoreDuplicates --extendReads'
SnakeMake DeepTools From line 82 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'macs2 callpeak -t {input.cellranger_bam} -g mm -f BAMPE -n {wildcards.sample} '
    '--outdir {params.macs_outdir} --keep-dup=1 --llocal 100000 --cutoff-analysis --min-length 1000 --max-gap 1000  2>&1 '
SnakeMake macs2 From line 93 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'macs2 callpeak -t {input.cellranger_bam} -g mm -f BAMPE -n {wildcards.sample} '
    '--outdir {params.macs_outdir} --keep-dup=1 --llocal {wildcards.llocal} 2>&1 '
SnakeMake macs2 From line 104 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'macs2 callpeak -t {input} -g mm -f BAMPE -n {wildcards.sample} '
    '--outdir {params.macs_outdir} --llocal 100000 --keep-dup=1 --broad-cutoff=0.1 ' 
    '--min-length 1000 --max-gap 1000 --broad 2>&1 '
SnakeMake macs2 From line 115 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    "wget -O {output} {params.url}"
SnakeMake From line 125 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    "bedtools genomecov -bg -g {input.genome} -i {input.fragments} > {output}"
SnakeMake BEDTools From line 134 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    "~/bin/SEACR/SEACR_1.3.sh {input} 0.01 norm relaxed {params.out_prefix}"
SnakeMake From line 144 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'python3 {params.script} {input.fragments} {wildcards.sample} | bgzip > {output.fragments}; '
    'tabix -p bed {output.fragments}'
SnakeMake tabix From line 155 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'bedtools intersect -abam {input.bam} -b {input.peaks} -u | samtools view -f2 | '
    'awk -f {params.get_cell_barcode} | sed "s/CB:Z://g" | python3 {params.add_sample_to_list} {wildcards.sample} | '
    'sort -T {params.tmpdir} | uniq -c > {output.overlap} && [[ -s {output.overlap} ]] ; '
SnakeMake SAMtools BEDTools From line 169 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
  ' samtools view -f2 {input.bam}| '
  'awk -f {params.get_cell_barcode} | sed "s/CB:Z://g" | python3 {params.add_sample_to_list} {wildcards.sample} | '
  'sort -T {params.tmpdir} | uniq -c > {output.all_bcd} && [[ -s {output.all_bcd} ]] ; '
SnakeMake SAMtools From line 183 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    "Rscript {params.script} --metadata {input.metadata} --fragments {input.fragments} --bcd_all {input.bcd_all} --bcd_peak {input.bcd_peak} --sample {wildcards.sample} --sample {wildcards.sample} --out_prefix {params.out_prefix}"
SnakeMake From line 205 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    "Rscript {input.script} --sample {wildcards.sample} --antibody {params.antibody} --metadata {input.metadata}  --fragments {input.fragments} --peaks {input.peaks} --out_prefix {params.out_prefix} --window {wildcards.binwidth} --genome_version {params.genome}"
SnakeMake From line 220 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'wget -O {output} {params.url}'
SnakeMake From line 228 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'cd {params.dirname}; '
    'tar -xvzf `basename {input.archive}`'
SnakeMake From line 238 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'wget -O {output.bw_tar} {params.url}'
SnakeMake From line 247 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    'cd {params.dirname}; '
    'tar -xvzf `basename {input.bw_tar}`'
SnakeMake From line 287 of workflow/Snakefile_pre_nbiotech.smk 
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shell:
    "python3 {input.script} -i {input.fastq} -o {params.out_folder} --single_cell --barcode {wildcards.barcode} 2>&1"
SnakeMake From line 64 of workflow/Snakefile_preprocess.smk 
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shell:
    'rm -rf results/multimodal_data/{wildcards.sample}/cellranger/{wildcards.sample}_{wildcards.antibody}_{wildcards.barcode}/; '
    'cd results/multimodal_data/{wildcards.sample}/cellranger/; '
    '/data/bin/cellranger-atac count --id {wildcards.sample}_{wildcards.antibody}_{wildcards.barcode} --reference {params.cellranger_ref} --fastqs {params.fastq_folder}'
SnakeMake From line 80 of workflow/Snakefile_preprocess.smk 
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shell:
    'bamCoverage -b {input.cellranger_bam} -o {output.bigwig} -p {threads} --minMappingQuality 5 '
    ' --binSize 50 --centerReads --smoothLength 250 --normalizeUsing RPKM --ignoreDuplicates --extendReads'
SnakeMake DeepTools From line 91 of workflow/Snakefile_preprocess.smk 
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shell:
    'macs2 callpeak -t {input.cellranger_bam} -g mm -f BAMPE -n {wildcards.antibody} '
    '--outdir {params.macs_outdir} --keep-dup=1 --llocal 100000 --cutoff-analysis --min-length 1000 --max-gap 1000  2>&1 '
SnakeMake macs2 From line 102 of workflow/Snakefile_preprocess.smk 
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shell:
    'macs2 callpeak -t {input.cellranger_bam} -g mm -f BAMPE -n {wildcards.antibody} '
    '--outdir {params.macs_outdir} --keep-dup=1 --llocal {wildcards.llocal} 2>&1 '
SnakeMake macs2 From line 113 of workflow/Snakefile_preprocess.smk 
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shell:
    'macs2 callpeak -t {input} -g mm -f BAMPE -n {wildcards.antibody} '
    '--outdir {params.macs_outdir} --llocal 100000 --keep-dup=1 --broad-cutoff=0.1 ' 
    '--min-length 1000 --max-gap 1000 --broad 2>&1 '
SnakeMake macs2 From line 124 of workflow/Snakefile_preprocess.smk 
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shell:
    'cut -f1,2 {params.faidx} > {output}'
SnakeMake From line 134 of workflow/Snakefile_preprocess.smk 
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shell:
    "bedtools genomecov -bg -g {input.genome} -i {input.fragments} > {output}"
SnakeMake BEDTools From line 143 of workflow/Snakefile_preprocess.smk 
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shell:
    "~/bin/SEACR/SEACR_1.3.sh {input} 0.01 norm relaxed {params.out_prefix}"
SnakeMake From line 153 of workflow/Snakefile_preprocess.smk 
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shell:
    'python3 {params.script} {input.fragments} {wildcards.sample} | bgzip > {output.fragments}; '
    'tabix -p bed {output.fragments}'
SnakeMake tabix From line 164 of workflow/Snakefile_preprocess.smk 
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shell:
    'bedtools intersect -abam {input.bam} -b {input.peaks} -u | samtools view -f2 | '
    'awk -f {params.get_cell_barcode} | sed "s/CB:Z://g" | python3 {params.add_sample_to_list} {wildcards.sample} | '
    'sort -T {params.tmpdir} | uniq -c > {output.overlap} && [[ -s {output.overlap} ]] ; '
SnakeMake SAMtools BEDTools From line 178 of workflow/Snakefile_preprocess.smk 
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shell:
  ' samtools view -f2 {input.bam}| '
  'awk -f {params.get_cell_barcode} | sed "s/CB:Z://g" | python3 {params.add_sample_to_list} {wildcards.sample} | '
  'sort -T {params.tmpdir} | uniq -c > {output.all_bcd} && [[ -s {output.all_bcd} ]] ; '
SnakeMake SAMtools From line 192 of workflow/Snakefile_preprocess.smk 
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shell:
    "Rscript {params.script} --metadata {input.metadata} --fragments {input.fragments} --bcd_all {input.bcd_all} --bcd_peak {input.bcd_peak} --antibody {wildcards.modality} --sample {wildcards.sample} --out_prefix {params.out_prefix}"
SnakeMake From line 214 of workflow/Snakefile_preprocess.smk 
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shell:
    "Rscript {input.script} --sample {wildcards.sample}   --antibody {wildcards.antibody} --metadata {input.metadata} --fragments {input.fragments} --out_prefix {params.out_prefix} --window {wildcards.binwidth} --genome_version {params.genome}"
SnakeMake From line 227 of workflow/Snakefile_preprocess.smk 
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shell:
    "Rscript {input.script} --sample {wildcards.sample}   --antibody {wildcards.modality} --metadata {input.metadata} --fragments {input.fragments} " \ 
    " --peaks {input.peaks} --out_prefix {params.out_prefix} --genome_version {params.genome}"
SnakeMake From line 242 of workflow/Snakefile_preprocess.smk 
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shell:
    'zcat {input.fragments} | sort -T {params.tmpdir} -k1,1 -k2,2n | bgzip > {output.fragments_merged} && tabix -p bed {output.fragments_merged}'
SnakeMake tabix From line 255 of workflow/Snakefile_preprocess.smk 
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shell:
    "bedToBam -i {input.fragments} -g {input.chrom_sizes} > {output.bam} && "
    "samtools sort -@ {threads} -o {output.bam_sorted} {output.bam} &&"
    "samtools index {output.bam_sorted} && "
    "bamCoverage -b {output.bam_sorted} -o {output.bigwig} -p {threads} --minMappingQuality 5 --binSize 50 --smoothLength 250 --normalizeUsing RPKM --ignoreDuplicates"
SnakeMake SAMtools DeepTools From line 268 of workflow/Snakefile_preprocess.smk 
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shell:
    'macs2 callpeak -t {input} -g mm -f BED -n {wildcards.modality} '
    '--outdir {params.macs_outdir} --llocal 100000 --keep-dup=1 --broad-cutoff=0.1 ' 
    '--min-length 1000 --max-gap 1000 --broad --nomodel 2>&1 '
SnakeMake macs2 From line 281 of workflow/Snakefile_preprocess.smk 
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shell:
    'Rscript {input.script} -i {input.seurat} -o {output.seurat}'
SnakeMake From line 293 of workflow/Snakefile_preprocess.smk 
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shell:
    'Rscript {params.script} -i {input.seurat} -o {output.seurat} -a {wildcards.feature} -d 40 -g {params.plot_group} '
SnakeMake From line 307 of workflow/Snakefile_preprocess.smk 
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shell:
    "Rscript {input.script} -i {input.seurat} -r {input.rna} -o {output}"
SnakeMake From line 54 of workflow/Snakefile_single_modality.smk 
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shell:
    "Rscript -e \"rmarkdown::render(input='{input.notebook}', "
    "                                output_file = '{params.report}', "
    "                                params=list(out_prefix = '{params.out_prefix}', "
    "                                           modality = '{wildcards.modality}', "
    "                                           feature = '{wildcards.feature}', "
    "                                           input = '{params.out_prefix}{input.seurat}', "
    "                                           integrated = '{params.out_prefix}{input.integrated}', "                # TODO - fix absolute paths integration                         
    "                                           output = '{params.out_prefix}{output.seurat}'))\" "
SnakeMake From line 68 of workflow/Snakefile_single_modality.smk 
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shell:
    'Rscript {input.script} --input {input.seurat} --output {output.seurat}'
SnakeMake From line 85 of workflow/Snakefile_single_modality.smk 
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shell:
    "Rscript {input.script}  --input {input.seurat} --fragments {input.fragments} --output_folder {output.bigwig} --idents {wildcards.idents} "
SnakeMake From line 95 of workflow/Snakefile_single_modality.smk 
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shell:
    "Rscript {input.script} -i {input.seurat} -o {output.markers} --idents {wildcards.idents}"
SnakeMake From line 105 of workflow/Snakefile_single_modality.smk 
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shell:
    'Rscript {input.script} -i {input.seurat} -o {output}'
SnakeMake From line 114 of workflow/Snakefile_single_modality.smk 
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shell:
    "Rscript {input.script} -i {input.seurat} -o {output.csv} -d {wildcards.ident}"
SnakeMake From line 123 of workflow/Snakefile_single_modality.smk 
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shell:
    "python3 {input.script} {input.bam} {wildcards.sample} {output.bam}"
SnakeMake From line 132 of workflow/Snakefile_single_modality.smk 
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shell:
    "samtools merge -@ {threads} {output.bam} {input.bam}"
SnakeMake SAMtools From line 151 of workflow/Snakefile_single_modality.smk 
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shell:
    'samtools index {input.bam}; '
    'bamCoverage -b {input.bam} -o {output.bw} -p {threads} --normalizeUsing RPKM'
SnakeMake SAMtools DeepTools From line 160 of workflow/Snakefile_single_modality.smk 
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shell:
    "python3 {input.script} {input.bam} {input.table} NA {output.bam_files}"
SnakeMake From line 172 of workflow/Snakefile_single_modality.smk 
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shell:
    'sh {input.script} {input.bam} {output.bw} {threads}'
SnakeMake From line 183 of workflow/Snakefile_single_modality.smk 
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shell:
    'cat {input.csv} | grep {wildcards.sample} > {output.csv_per_sample}; '
    'python3 {input.script} {input.bam} {output.csv_per_sample} NA {output.bam_per_cluster}'
SnakeMake From line 194 of workflow/Snakefile_single_modality.smk 
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shell:
    'sh {input.script} {input.bam} {output.bw} {threads}'
SnakeMake From line 205 of workflow/Snakefile_single_modality.smk 
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shell:
    'sh {input.script}  {input.bam} {output.peaks}'
SnakeMake From line 214 of workflow/Snakefile_single_modality.smk 
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shell:
    'Rscript {input.script} --input {input.csv} --nmarkers {params.nmarkers} --output {output.bed}'
SnakeMake From line 225 of workflow/Snakefile_single_modality.smk 
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shell:
    'python3 {params.script} {input} > {output}'
SnakeMake From line 236 of workflow/Snakefile_single_modality.smk
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://github.com/mardzix/bcd_nano_CUTnTag
Name: bcd_nano_cutntag
Version: 1
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