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3D structure of the Neurospora crassa genome at 50 kb resolution

3D genome builder (3DGB)

3D genome builder (3DGB) is a workflow to build 3D models of genomes from HiC raw data and to integrate omics data on the produced models for further visual exploration. 3DGB bundles HiC-Pro, PASTIS and custom Python scripts into a unified Snakemake workflow with limited inputs (see Preparing Required Files). 3DGB produces annotated 3D models of genome in PDB and G3D formats.

SWH

Download this repository

git clone https://github.com/data-fun/3d-genome-builder.git
cd 3d-genome-builder

Install dependencies

Singularity

Download the latest version here

Install Singularity:

sudo apt install -y ./singularity-container_3.8.7_amd64.deb

Verify version:

$ singularity --version
singularity version 3.8.7

Conda environment

Install conda.

Install mamba:

conda install mamba -n base -c conda-forge

Create conda environment and install dependendies:

mamba env create -f binder/environment.yml

Load conda environment:

conda activate 3DGB

Download HiC-Pro Singularity image

wget --ciphers=DEFAULT:@SECLEVEL=1 https://zerkalo.curie.fr/partage/HiC-Pro/hicpro_3.1.0_ubuntu.img -P images

If this command fails, try with an alternate download link:

wget https://zenodo.org/record/8376626/files/hicpro_3.1.0_ubuntu.img -P images

Check the integrity of the image:

$ md5sum images/hicpro_3.1.0_ubuntu.img
d480e636397c14e187608e50309eb9af  images/hicpro_3.1.0_ubuntu.img

Verify HiC-Pro version with:

$ singularity exec images/hicpro_3.1.0_ubuntu.img HiC-Pro --version
[...]
HiC-Pro version 3.1.0

and bowtie2 version:

$ singularity exec images/hicpro_3.1.0_ubuntu.img bowtie2 --version  2>/dev/null | head -n 1
/usr/local/conda/envs/hicpro/bin/bowtie2-align-s version 2.4.4

Prepare required files

Create the config file

Create and edit a configuration file in yaml format. See for instance the template config_template.yml

Add the reference genome

The reference genome fasta file must be located in WORKING_DIR/genome.fasta where WORKING_DIR is the name of the working directory as specified in your config file.

Add FASTQ files (optional)

If you already have fastq files stored locally or some fastq files are not available on GEO or SRA, you can use these files providing they are in the proper directory structure:

3D structure of the chromosome 13 of S. cerevisiae at 5 kb resolution

WORKING_DIR/
├── fastq_files
│   ├── ID1
│   │   ├── ID1_R1.fastq.gz
│   │   └── ID1_R2.fastq.gz
│   ├── ID2
│   │   ├── ID2_R1.fastq.gz
│   │   └── ID2_R2.fastq.gz
│   ├── ID3
│   │   ├── ID3_R1.fastq.gz
│   │   └── ID3_R2.fastq.gz
│   └── ID4
│       ├── ID4_R1.fastq.gz
│       └── ID4_R2.fastq.gz
└── genome.fasta
  • WORKING_DIR is the name of the working directory as specified in your config file.
  • Paired-end fastq files are in the directory WORKING_DIR/fastq_files/IDx with IDx the identifier of the paired fastq files. Fastq identifiers are reported in the config file. Please note fastq files have to follow the pattern <sample ID>_R<1 or 2>.fastq.gz.

Note

Please strictly follow this file organization as it is required by the 3DGB workflow.

Build model

Run 3DGB:

snakemake --profile smk_profile -j 4 --configfile YOUR-CONFIG.yml

Note

  • Adapt YOUR-CONFIG.yml to the exact name of the config file you created.
  • Option -j 4 tells Snakemake to use up to 4 cores. If you are more cores available, you can increase this value (e.g. -j 16).

Or with debugging options:

snakemake --profile smk_profile_debug -j 4 --configfile YOUR-CONFIG.yml --verbose

Depending on the number and size of fastq files, the 3D construction will take a couple of hours to run.

For troubleshooting, have a look to log files in WORKING_DIR/logs, where WORKING_DIR is the name of the working directory as specified in your config file.

Map quantitative values on the 3D model

To map quantitative values on the model run:

python ./scripts/map_parameter.py --pdb path/to/structure.pdb --bedgraph path/to/annotation.bedgraph --output path/to/output.pdb

Quantitative values should be formatted in a 4-column bedgraph file (chromosome/start/stop/value):

chr1	0	50000	116.959
chr1	50000	100000	48.4495
chr1	100000	150000	22.8726
chr1	150000	200000	84.3106
chr1	200000	250000	113.109

Each bead of the model will be assigned a quantitative value. The resolution in the bedgraph file should match the resolution used to build the model.

Get results

Upon completion, the WORKING_DIR should look like this:

WORKING_DIR/
├── contact_maps
├── dense_matrix
├── fastq_files
├── HiC-Pro
├── logs
├── pastis
├── sequence
└── structure

The following paths contain the most interesting results:

  • WORKING_DIR/contact_maps/*.png : contact maps.
  • WORKING_DIR/HiC-Pro/output/hic_results/pic/*/*.pdf : graphical summaries of read alignments produced by Hi-C Pro.
  • WORKING_DIR/pastis/structure_RESOLUTION.pdb : raw 3D models (in PDB format) produced by Pastis.
  • WORKING_DIR/structure/RESOLUTION/structure_cleaned.* : final (annotated) 3D models in PDB and G3D formats.

Note

  • WORKING_DIR is the name of the working directory as specified in your config file.
  • RESOLUTION is the resolution of the Hi-C data specified in the config file.

Examples

Visualize 3D model structures

To visualize 3D model structures (.pdb and .g3d files), follow this quick tutorial.

Build DAG graph

For visualization purpose, you can build the graph of all computational steps involved in the 3D construction of the genome.

snakemake --profile smk_profile --configfile YOUR-CONFIG.yml --rulegraph  | dot -Tpdf > rules.pdf

where YOUR-CONFIG.yml should be replaced by the name of the config file you created.

With wildcards:

snakemake --profile smk_profile --configfile YOUR-CONFIG.yml --dag  | dot -Tpdf > dag.pdf