ChIP-req read allocation for multiple alignments

This repo contains Snakemake pipelines to align ChIP-seq data with multiple mapping followed by CSEM (Chung et al. 2011) allocation. These pipelines were developed for the following manuscript:

Diverse molecular mechanisms contribute to differential expression of human duplicated genes. Shew CJ, Carmona-Mora P, Soto DC, Mastoras M, Roberts E, Rosas J, Jagannathan D, Kaya G, O'Geen H, and Dennis MY. MBE. 2021.

Snakefile_shortread: used for analysis of short ENCODE reads (single-end)

Snakefile_long: used for analysis of large-insert ChIP libraries (paired-end)

1) Required software

Besides snakemake, this pipeline uses the following tools:

• fastqc
• trimmomatic
• samtools
• bowtie (short read) bowtie2 (long read)
• macs2
• csem
• rsem (long chip pipeline only)
• bedGraphToBigWig
• phantompeakqualtools

Most tools are automatically installed by snakemake through Conda. CSEM and BedGraphToBigWig must be installed locally (they are not yet available in Conda repositories).

2) Folder structure

The following structure is recommended:

|__ adapters
    |__ sequences.fa 
|__ config.yaml # Mandatory: described in section 3
|__ reads 
    |__ sample1.fastq 
    |__ sample2.fastq
    |__ control1.fastq
    |__ ...
|__ envs
    |__ fastqc.yaml
    |__ macs2.yaml
    |__ samtools.yaml
    |__ trimmomatic.yaml
|__ readme.md
|__ reference
    |__ masked_reference.fa
    |__ chromosome_size.txt
|__ scripts
    |__ sampling.sh or long_sampling.sh
|__ Snakefile

The pipeline will generate results directory containing intermediate files and peaks called.

3) Config file

Config file must contain the following information:

# Analysis name

filename: example

# Input files

samples:
  - sample1
  - sample2
  - ...

control:
  - control1
  - ...
  
samples_size: # from phantompeakqualtools
  - 485
  - 485

control_size: # from phantompeakqualtools
  - 665
  - 655

adapters: adapters/sequences.fa

# Reference path

reference: reference/masked_reference.fa
chromsize: reference/chromosome_sizes.txt

# Peak calling options

broad: boolean

# Workflow options

csem: boolean

In order to obtain the values for samples_size and control_size in the config file, we need to predict fragment size for each dataset using the alignments obtained with bwt-default and PhantomPeakQualTools. To do this, we need to run the Snakefile until sam2bam rule and then run phantompeaktools. After obtaining the sizes from phantompeaktools, update the samples_size and control_size values in the config file and then run the full snakemake pipeline as described in Section 4.

Obtaining sizes example:

snakemake -s Snakefile --configfile GM12878_H3K27ac.yaml -p -j 10 --until sam2bam
Rscript /share/dennislab/programs/phantompeakqualtools/run_spp.R -c=results/alignments/ENCFF001CUR.trimmed.bwt-df.bam

4) Execution

Export locally installed software to your PATH:

export PATH="/share/dennislab/programs/share_path:/share/dennislab/programs/csem-2.4:$PATH"

Dry-run:

snakemake --configfile config.yaml --use-conda -n

Obtain workflow graphs:

snakemake --configfile config.yaml --dag | dot -Tpng > dag.png 
snakemake --configfile config.yaml --rulegraph | dot -Tpng > rulegraph.png

Run interactively:

snakemake --configfile config.yaml --use-conda -j 20 -p