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HiC-Pro

Introduction

Hi-C data processing pipeline using HiC-Pro.

Applied

In practice, HiC-Pro was successfully applied to many data-sets including dilution Hi-C, in situ Hi-C, DNase Hi-C, Micro-C, capture-C, capture Hi-C or HiChip data.

Require Software

The HiC-Pro pipeline requires the following dependencies :

  • The bowtie2 mapper
  • Python (>2.7) with pysam (>=0.8.3), bx-python(>=0.5.0), numpy(>=1.8.2), and scipy(>=0.15.1) libraries. Note that the current version does not support python 3
  • R with the RColorBrewer and ggplot2 (>2.2.1) packages
  • g++ compiler
  • samtools (>1.1)
  • homer

Install (in a dependent environment)

Create a new environment

$ conda create -n py2 python=2.7
$ source activate py2

Dependencies install

$ conda install bowtie2
$ conda install python=2.7
$ conda install pysam
$ conda install bx-python
$ conda install numpy
$ conda install scipy 
$ conda install R
$ conda install r-ggplot2
$ conda install r-rcolorbrewer
$ conda install samtools
$ conda install homer

HiC-Pro install

$ wget https://github.com/zhengzhanye/HiC-Pro/archive/master.zip
$ unzip master.zip
$ cd HiC-Pro-master
Edit configure-install.txt (if dependencies install successful,you only need manually defined the path to  'installation folder'; else you need edit the config-install.txt file and manually defined the paths to dependencies.)
$ make configure
$ make install

Annotation Files

  • A table file of chromosomes' size.

    $ mkdir 00_hg19
$ cd 00_hg19
$ wget https://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/hg19.fa.gz
$ gunzip hg19.fa.gz
$ http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/hg19.chrom.sizes
$ head -n 24 hg19.chrom.sizes | sort -V > ./hg19_size.txt

  • A BED file of the restriction fragments after digestion.This file depends both of the restriction enzyme and the reference genome.

       chr1   0       16007   HIC_chr1_1    0   +
   chr1   16007   24571   HIC_chr1_2    0   +
   chr1   24571   27981   HIC_chr1_3    0   +
   chr1   27981   30429   HIC_chr1_4    0   +
   chr1   30429   32153   HIC_chr1_5    0   +
   chr1   32153   32774   HIC_chr1_6    0   +
   chr1   32774   37752   HIC_chr1_7    0   +
   chr1   37752   38369   HIC_chr1_8    0   +
   chr1   38369   38791   HIC_chr1_9    0   +
   chr1   38791   39255   HIC_chr1_10   0   +
   (...)

    NOTE: HindIII_resfrag_hg19.bed and HindIII_resfrag_mm10.bed has been provides in annotation dir.

    ## For example：生成MboI 限制性内切酶 bed 文件，参考基因组hg19
## -r 指定酶切位点 （）
## -o 指定输出文件名称
$ $PATH_of_hic/HiC-Pro_2.11.1/bin/utils/digest_genome.py -r ^GATC -o hg19_mobi.bed /$path_of_hg19/hg19.fa

  • The bowtie2 indexes.

    $ cd 00_hg19
$ mkdir bowtie2
$ bowtie2-build --threads 30 -f /hg19.fa ./bowtie2/hg19

Usage

  1. uniform data formate

    $ cd bin
$ perl 01.0_pre4hicpro.pl

    detail information see 01.0_pre4hicpro.pl.

  2. run hic-pro

    $ bash 01.1_hicpro.sh

    NOTE： 需要修改 .sh 中的HiC-Pro的路径，以及修改 $fcfg指代的cfg 文件中的路径。

  3. bam to sam

    $ bash 02.0_bam2sam.sh

  4. homer call loop

    $ bash 03.0_HOME.sh

  5. Deactivate environment

    $ source deactivate py2

Troubleshooting

  1. 文件太大时，sort时会产生很多临时文件，系统一直处于写的状态，此时就 会报错（samtools sort: fail to open "tmp/H1-hESC_DpnII_inSitu_1_hg19.1020.bam": Too many open files）

    method:

    $path/HiC-Pro_2.11.1/scripts/bowtie_combine.sh 文件中sort 命令（## Sort merge file.版块）在sort -@ 中间加-m 10G 变成 sort -m 10G -@

Reference

https://github.com/nservant/HiC-Pro

http://bowtie-bio.sourceforge.net/bowtie2/index.shtml