hic-pipeline

A pipeline for scaffolding a genome assembly using Hi-C and SALSA2, generalizable to whatever job scheduler or local system you are using. Loosely based on the Arima Hi-C mapping pipeline.

Options for running

I use nextflow to run this pipeline, because it automatically deals with submitting cluster jobs and containerization. You can run it manually too, though, or write your own scripts. There is a section below for both of these options.

Requirements

If you're not using docker or singularity, you'll need the following software installed:

bwa (tested with 0.7)
samtools (tested with 1.9)
bedtools (tested with 2.26)
python3 and pysam
SALSA2 (requires python2.7)

No installation is necessary for this pipeline; just download it and put the resulting folder in your path:

git clone https://github.com/esrice/slurm-hic.git
export PATH=$PATH:$(PWD)/slurm-hic

Running manually

Mapping reads

First, index your contigs if you haven't already:

bwa index contigs.fa
samtools faidx contigs.fa

Now, align the R1 and R2 reads to your contigs separately, filtering the output through the filter_chimeras.py script to remove experimental artifacts from the alignments:

bwa mem contigs.fa HiC-reads_R1.fastq.gz | samtools view -bh - \
    | filter-chimeras.py - > r1.bam
bwa mem contigs.fa HiC-reads_R2.fastq.gz | samtools view -bh - \
    | filter-chimeras.py - > r2.bam

Combining paired reads

Combine the r1 and r2 files using combine_ends.py, and then fix mates, sort, and remove PCR duplicates:

combine_ends.py r1.bam r2.bam | samtools fixmate -m - - | samtools sort - \
    | samtools markdup -r - combined.bam

Run SALSA2

SALSA2 takes alignments in bed rather than bam format, sorted by read name. Here's the command to do that:

bamToBed -i combined.bam | sort -k 4 > combined.bed

Now, just run SALSA. This is the command I use, but see the manual for more options:

python2.7 run_pipeline.py -a contigs.fa -l contigs.fa.fai -b combined.bed \
    -e GATC -m yes -o salsa -p yes

Running using nextflow

You can also run this pipeline with a single command using nextflow. If you have a way to run commands inside containers, such as singularity or docker, you don't even need to worry about installing requirements! Nextflow is easy to install:

wget -qO- https://get.nextflow.io | bash

This command creates the nextflow binary in the current directory. You can put it somewhere in your $PATH if you want.

Configuring nextflow

Nextflow looks for the file nextflow.config in the current directory for instructions on how to run the pipeline, e.g., whether to run it locally or using a cluster scheduler. You can read the nextflow documention on configuration for more information, but here's what I use to run the pipeline on SLURM:

process {
    executor = 'slurm'
    queue = 'BioCompute'
    time = '2d'
    memory = '48 GB'
}

Nextflow also supports SGE, LSF, PBS, AWS, and several other batch systems.

Configuring to use locally installed software

Make sure the software specified in the Requirements section is installed and in your path. If you're in an HPC/cluster environment that uses the module system for software, make sure to tell nextflow to load the correct modules, e.g., by putting the following line in nextflow.config:

process.module = 'bwa/0.7:samtools/1.9:bedtools/2.26'

Also, let nextflow know where the SALSA2 python files live:

env.SALSA_PATH = '/path/to/SALSA'

Configuring to use containers

If you can use docker or singularity, you don't need to worry about installing any requirements. Just add the following lines to nextflow.config:

process.container = 'esrice/hic-pipeline:latest'
singularity.enabled = true

substituting docker for singularity if using docker. Depending on how you have singularity set up, you may need to add singularity.autoMount = true or singularity.runOptions = "--bind /storage" where /storage is the root of your data filesystem. See nextflow documentation on docker and singularity for more information.

Running the pipeline

Just run the following command:

nextflow run hic-pipeline.nf --reference contigs.fa \
    --r1_reads HiC-reads_R1.fastq.gz --r2_reads HiC-reads_R2.fastq.gz \
    --enzyme GATC

The output will be in salsa_out/. If the pipeline gets cut off before finishing, you can start from where it left off by adding the -resume flag.

amvarani / hic-pipeline