satyanarayan-rao/star_protocol_enhancer_cooperativity

STAR protocol for cooperative binding analysis using dSMF data

This protocol is derived from Rao et al., 2021.

Before you begin

Download the pipeline

Method 1:

If git command is available on the machine you want to run the pipeline, it can simply be downlaod using the following command:

git clone https://github.com/satyanarayan-rao/star_protocol_enhancer_cooperativity.git

Method 2

Please visit the github repository here. Please click on the code and choose "Download Zip" option as shown in the image below.

Install required softwares

This pipeline is Linux/Unix-based system compatible.

Please install Anaconda Individual Edition first.

Please follow the steps below to build right environment to run the pipeline.

• Create an environment dsmf_viz using the command: conda create -n dsmf_viz python=3.6
• Activate this this environment using command source activate dsmf_viz
• Run install_required_packages.sh to install required packages mentioned below:
  • Bowtie2
  • Bismark
  • Trim Galore
  • Snakemake
  • Bedtools
  • Samtools
  • Bamtools
  • pyBigWig
  • Pandas
  • Numpy
  • Tbb
  • Gnuplot
  • Ghostscript
  • Perl

CAUTION: Please run install_required_packages.sh only after activating the virtual environment (dsmf_viz) to avoid conflicts with existing package installations

Download reference genome and dSMF data

Please run the following command to download dm3 reference genome.

$ sh download_reference_genome.sh

Data for demo is included in this github repository, but to visualize at your sites of interest, please download the sequencing data, and keep them in data_from_geo/. Here is the list of URLs for the sequencing data.

ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR313/006/SRR3133326/SRR3133326_1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR313/006/SRR3133326/SRR3133326_2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR313/007/SRR3133327/SRR3133327_1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR313/007/SRR3133327/SRR3133327_2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR313/008/SRR3133328/SRR3133328_1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR313/008/SRR3133328/SRR3133328_2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR313/009/SRR3133329/SRR3133329_1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR313/009/SRR3133329/SRR3133329_2.fastq.gz

Directory structure

• configs/: contains configuration file for the pipeline. Please see the exmaple demo_S2 in configs/config.yaml to add your own sample information. configs/cluster.json contains information for submitting jobs on cluster. Plese contact your cluster system administrator to configure this json file accordingly.

• input_bed/: Here user should keep regions of interest in a bed file. Please look at input_bed/example.bed for mapping binding at single sites, and see input_bed/example_cobinding.bedpe for mapping binding at pair of sites.

• data_from_geo/: This directory contains raw sequencing reads

• ref_genome/: This directory contains reference genome of your interest

• metadata/: This directory contains meta information, for example, genome size file, metadata/dm3.chrom.sizes. Please use appropriate genome size correspoding to the reference genome!

• plots/: Contains subdirectories with output pdf visualizing footprints and methylation maps

• utils/gnuplot_base_files/: Contains gnuplot commands in files that are used while plotting

• scripts/: Contains required scripts to run the pipeline

• snakemakes/: Contains modularized snakemake files. File names are self-explanatory

• workflow_figures/: Contains snakemake workflow image. Names of rules in the image can be traced in the snakemake files

Run the pipeline

To reproduce panels of Figure1 in the STAR protocol manuscript

Please run the following single command.

snakemake --snakefile cooperative_binding_analysis.smk plots/single_binding/suppressed_merged_demo_S2_to_example_spanning_lf_15_rf_15_extended_left_150_right_150_roi_peak_229.fp.pdf plots/single_binding/suppressed_merged_demo_S2_to_example_spanning_lf_15_rf_15_extended_left_150_right_150_roi_peak_229.methylation.pdf --configfile configs/config.yaml

To reproduce panels of Figure2 in the STAR protocol manuscript

snakemake  --snakefile cooperative_binding_analysis.smk plots/cobinding_bedpe/suppressed_merged_demo_S2_to_example_cobinding_lf_15_rf_15_extended_left_300_right_300_roi_peak_110_4_and_peak_110_6.fp.pdf plots/cobinding_bedpe/suppressed_merged_demo_S2_to_example_cobinding_lf_15_rf_15_extended_left_300_right_300_roi_peak_110_4_and_peak_110_6.methylation.pdf --configfile configs/config.yaml

Interpreting file names:

The advantage of Snakemake is that a user can incorporate parameters in file names. Related to this, below I expand on parameters placed in the output file names:

For a single binding site example

File name: plots/single_binding/suppressed_merged_demo_S2_to_example_spanning_lf_15_rf_15_extended_left_150_right_150_roi_peak_229.fp.pdf

• demo_S2: points to the samples. Please take a look at samples starting with demo_S2 in data_from_geo/samples.tsv and also look at bam_merge_config -> demo_S2 in configs/config.yaml file

• example: points to input_bed/example.bed

• 15: span 15bp from the ROI center; lf means span left, and rf means span right. This parameter is used in defining TF footprint.

• 150: span 150 bp from ROI center. This is for visualization purpose. A dSMF molecule in principle could be as long as 300 bp, thus spanning 150 bp left and right respectively.

• peak_229: Name of the ROI. This name can be found as the fourth column in input_bed/example.bed

For a pair of binding sites example

File name: plots/cobinding_bedpe/suppressed_merged_demo_S2_to_example_cobinding_lf_15_rf_15_extended_left_300_right_300_roi_peak_110_4_and_peak_110_6.fp.pdf

• demo_S2: Same as above

• example_cobinding: points to input_bed/example_cobinding.bedpe ; CRITICAL: the file name should have .bedpe extension and should follow bedpe format.

• 15: same as above: this parameter will be used for defining TF footprints at both ROIs

• 300: span 300bp from the left ROI (Chromosom location of ROI_left < ROI_right)

• peak_110_4_and_peak_110_6: name_of_left_ROIandname_of_right_ROI; this name can be found in input_bed/example_cobinding.bedpe