We recommend using conda to run HiCzin.
After installing Anaconda (or miniconda), Users can clone the repository with git:
git clone https://github.com/dyxstat/HiCzin.git
Once complete, you can enter the repository folder and then create a HiCzin environment using conda:
# enter the HiCzin folder
cd HiCzin
# Construct environment
conda env create -f HiCzin_linux_env.yaml
or
conda env create -f HiCzin_osx_env.yaml
# Enter the environment
conda activate HiCzin_env
Normalization method in HiCzin depends on R package 'glmmTMB'. Though the R package can be installed by 'conda install -c conda-forge r-glmmtmb', you will meet one potential warning derived from the dependency version (glmmTMB/glmmTMB#615): 'Error in .Call("FreeADFunObject", ptr, PACKAGE = DLL) : "FreeADFunObject" not available for .Call() for package "glmmTMB"' and we are not sure whether this warning would influence the noramlization results. To get rid of this warning, we strongly recommend you to install the source version of package 'glmmTMB' directly in R:
# Enter the R
R
# Download the R package and you may need to select a CRAN mirror for the installation
install.packages("glmmTMB", type="source")
Finally, you can test the pipeline, and testing result are in test/out/hiczin.log:
python ./hiczin.py test test/out
For the shotgun library, de novo metagenome assembly is produced by an assembly software, such as MEGAHIT.
megahit -1 SG1.fastq.gz -2 SG2.fastq.gz -o ASSEMBLY --min-contig-len 1000 --k-min 21 --k-max 141 --k-step 12 --merge-level 20,0.95
Hi-C paired-end reads are mapped to assembled contigs using BWA-MEM with parameters ‘-5SP’. Then, samtools with parameters ‘view -F 0x904’ is applied to remove unmapped reads (0x4) and supplementary (0x800) and secondary (0x100) alignments and sort BAM files by name.
bwa index final.contigs.fa
bwa mem -5SP final.contigs.fa hic_read1.fastq.gz hic_read2.fastq.gz > MAP.sam
samtools view -F 0x904 -bS MAP.sam > MAP_UNSORTED.bam
samtools sort -n MAP_UNSORTED.bam -o MAP_SORTED.bam
Firstly, BBmap from the BBTools suite is applied to map the shotgun reads to the assembled contigs with parameters ‘bamscript=bs.sh; sh bs.sh’. The coverage of contigs is computed using script: ‘jgi summarize bam contig depths’ from MetaBAT2 v2.12.1.
bbmap.sh in1=SG1.fastq.gz in2=SG2.fastq.gz ref=final.contigs.fa out=SG_map.sam bamscript=bs.sh; sh bs.sh
jgi_summarize_bam_contig_depths --outputDepth coverage.txt --pairedContigs pair.txt SG_map_sorted.bam
python ./hiczin.py norm [Parameters] FASTA_file BAM_file COV_file OUTPUT_directory
-e: Case-sensitive enzyme name. Use multiple times for multiple enzymes
(Optional; If no enzyme is input, HiCzin_LC mode will be automatically employed to do normalization)
--min-len: Minimum acceptable contig length (default 1000)
--min-mapq: Minimum acceptable alignment quality (default 30)
--min-match: Accepted alignments must be at least N matches (default 30)
--min-signal: Minimum acceptable signal (default 2)
--thres: Maximum acceptable fraction of incorrectly identified valid contacts in spurious contact detection (default 0.05)
-v: Verbose output
- FASTA_file: a fasta file of the assembled contig (e.g. final.contigs.fa)
- BAM_file: a bam file of the Hi-C alignment (e.g. MAP_SORTED.bam)
- COV_file: a txt file of contigs' coverage information computed by script: ‘jgi summarize bam contig depths’ from MetaBAT2 (e.g. depth.txt)
python ./hiczin.py norm -e HindIII -e NcoI -v final.contigs.fa MAP_SORTED.bam depth.txt out
All outputs of HiCzin normalization pipeline are located in the OUT_directory you specified when running the software.
- hiczin.log: the specific implementation information of HiCzin pipeline
- contig_info.csv: information of contigs, containing four columns (contigs' name, the number of restriction sites on contigs, contigs' length and coverage) or three columns (contigs' name, length and coverage)
- Normalized_contact_matrix.npz: a sparse matrix of normlized Hi-C contact maps in csr format and can be reloaded using scipy.sparse.load_npz('Normalized_contact_matrix.npz')
- valid_contact.csv: information of valid contacts, containing three columns (index of the first contig, index of the second contig, and value of raw valid intra-species contacts)
- HiCzin_normalized_contact.gz: Compressed format of the normalized contacts and contig information by pickle. This file can further serve as the input of HiCBin and HiFine.
If you have any questions or suggestions, welcome to contact Yuxuan Du (yuxuandu@usc.edu).
This program is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version.
This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.
You should have received a copy of the GNU General Public License along with this program. If not, see http://www.gnu.org/licenses/.