Identify real loops from Hi-C data.
hicpeaks provide a Python CPU-based implementation for BH-FDR and HICCUPS, two peak calling algorithms for Hi-C data, proposed by Rao et al .
hicpeaks is developed and tested on UNIX-like operating system, and following packages or softwares are required:
- Python 2.7/3.5+
conda, an excellent package manager, can be used to install all requirements above.
If you have the Anaconda Distribution installed, you already have it.
Download the latest Linux Miniconda installer, then in your terminal window type the following and follow the prompts on the installer screens:
$ bash Miniconda2-latest-Linux-x86_64.sh
After that, update the environment variables to finish the Conda installation:
$ source ~/.bashrc
Install Packages through Conda
Conda allows separation of packages into separate repositories, or channels. The main defaults channel has a large amount of common packages including numpy, scipy, pandas, statsmodels, scikit-learn, and h5py listed above. To install these packages, type and execute the following command:
$ conda install numpy scipy matplotlib pandas statsmodels scikit-learn h5py
Other packages: cooler and ucsc-fetchchromsizes are not available in the defaults channel but included in the bioconda channel, and multiprocess is included in the conda-forge channel. To make them accessible, you need to add the bioconda channel as well as the other channels bioconda depends on (note that the order is important to guarantee the correct priority):
$ conda config --add channels conda-forge $ conda config --add channels defaults $ conda config --add channels r $ conda config --add channels bioconda
To install these requirements:
$ conda install multiprocess cooler ucsc-fetchchromsizes
Now just download the hicpeaks source code from PyPI, extract it and run the setup.py script:
$ python setup.py install
hicpeaks would be installed successfully if no exception occurs in the above process.
hicpeaks comes with 4 scripts: toCooler, pyBHFDR, pyHICCUPS and peak-plot.
Store TXT/NPZ bin-level Hi-C data into cooler container.
- I have included a sample data with hicpeaks source code to illustrate how you should prepare your data in TXT format. It’s quite easy, just remember 3 points: 1. the file name should follow this pattern “chrom1_chrom2.txt” (remove prefix from your chromosome labels, i.e. “chr1” should be “1”, and “chrX” should be “X”); 2. each file should only contain 3 columns, corresponding to “bin1” of “chrom1”, “bin2” of “chrom2”, and “contact frequency” (don’t perform any normalization processes); 3. all files at the same resolution should be placed under a single folder.
- NPZ format is another bin-level Hi-C data container which can extremely speed up data loading. hicpeaks supports NPZ files generated by runHiC and TADLib.
A CPU-based python implementation for BH-FDR algorithm. Rao et al states in their supplementary material that this algorithm is robust enough to obtain all main results of their paper. Compared with HICCUPS, BH-FDR doesn’t use λ-chunk in multiple hypothesis test, and only considers the Donut background region when calculating the expected values.
A CPU-based python implementation for HICCUPS algorithm. Besides the donut region, HICCUPS also considers the lower-left, vertical and horizontal backgrounds when calculating the expected values. And λ-chunk is used to overcome several multiple hypothesis testing challenges for Hi-C data. Finally, while BH-FDR has to limit the detected pixels near the diagonal (<2Mb), HICCUPS is able to generalize itself to any genomic distance in theory. Here, pyHICCUPS keeps all main concepts of the original algorithm except for these points which may be fixed in the near future:
- pyHICCUPS excludes vertical and horizontal backgrounds from its calculation.
- pyHICCUPS cannot combine peak annotations at different resolutions.
- Due to computational complexity, you should still limit the genomic distance of 2 loci to some degree (5Mb/10Mb).
Although these differences, peaks returned by pyHICCUPS are quite consistent with our visual inspection, and generally follow the typical loop interaction patterns.
Visualize peaks (or loops) detected by pyBHFDR or pyHICCUPS on heatmap. Just provide a cooler file and a loop annotation file, and input your interested region (chrom, start, end), peak-plot will export the figure in PNG format.
This tutorial will guide you through the basic usage of all scripts distributed with hicpeaks.
If you have already created a cooler file for your Hi-C data, skip to the next section pyBHFDR and pyHICCUPS, go on otherwise.
First, you should store your TXT/NPZ bin-level Hi-C data into a cooler file by using toCooler. Let’s begin with our sample data below. Suppose you are still in the hicpeaks distribution root folder: change your current working directory to the sub-folder example:
$ cd example $ ls -lh * -rw-r--r-- 1 xtwang staff 18B Aug 21 19:46 datasets -rw-r--r-- 1 xtwang staff 293B Aug 23 20:53 hg38.chromsizes 40K: total 11608 -rw-r--r-- 1 xtwang staff 2.7M Aug 21 19:44 21_21.txt -rw-r--r-- 1 xtwang staff 2.9M Aug 21 19:44 22_22.txt
There are one sub-directory called 40K which contains Hi-C data of two chromosomes in K562 cell line at 40K resolution, and one metadata file datasets which we can pass directly to toCooler:
$ cd 40K $ head -5 21_21.txt 250 251 1 250 258 1 250 259 1 250 260 4 250 261 2 $ cd .. $ cat datasets res:40000 ./40K
You should construct your TXT files (no head, no tail) with 3 columns, which indicate “bin1 of the 1st chromosome”, “bin2 of the 2nd chromosome” and “contact frequency” respectively. See Overview above.
To transform this data to cooler format, just run the command below:
$ toCooler -O K562-MboI-parts.cool -d datasets --assembly hg38 --nproc 2
toCooler routinely fetch sizes of each chromosome from UCSC with the provided genome assembly name (here hg38). However, if your reference genome is not holded in UCSC, you can also build a file like “hg38.chromsizes” in current working directory, and pass the file path to the argument “–chromsizes-file”.
Type toCooler with no arguments on your terminal to print detailed help information for each parameter.
For this datasets, toCooler will create a cooler file named “K562-MboI-parts.cool”, and your data will be stored under the URI “K562-MboI-parts.cool::40000”.
This tutorial only illustrates a very simple case, in fact the metadata file may contain list of resolutions (if you have data at different resolutions in the same cell line) and corresponding folder paths (both relative and absolute path are accepted, and if your data are NPZ format, this path should point to the NPZ file):
res:10000 /absoultepath/10K res:20000 ../relativepath/20K res:40000 /npzfile/anyprefix.npz
Then toCooler will generate a single cooler file storing all the specified data under different cooler URI: “specified_cooler_path::10000”, “specified_cooler_path::20000” and “specified_cooler_path::40000”.
pyBHFDR and pyHICCUPS
With cooler URI, you can perform peak annotation by pyBHFDR or pyHICCUPS:
$ pyBHFDR -O K562-MboI-BHFDR-loops.txt -p K562-MboI-parts.cool::40000 -C 21 22 --pw 1 --ww 3
$ pyHICCUPS -O K562-MboI-HICCUPS-loops.txt -p K562-MboI-parts.cool::40000 --pw 1 2 4 --ww 3 5 7
pyHICCUPS supports multiple parameters since 0.3.0, in which case it will combine peak annotations from different parameter settings automatically.
Type pyBHFDR or pyHICCUPS on your terminal to print detailed help information for each parameter.
Before step to the next section, let’s list the contents under current working directory again:
$ ls -lh total 1744 drwxr-xr-x 5 xtwang staff 160B Sep 3 14:55 40K -rw-r--r-- 1 xtwang staff 3.9K Sep 3 14:58 BHFDR.log -rw-r--r-- 1 xtwang staff 17K Sep 3 14:59 HICCUPS.log -rw-r--r-- 1 xtwang staff 16K Sep 3 14:58 K562-MboI-BHFDR-loops.txt -rw-r--r-- 1 xtwang staff 19K Sep 3 14:59 K562-MboI-HICCUPS-loops.txt -rw-r--r-- 1 xtwang staff 704K Sep 3 14:57 K562-MboI-parts.cool -rw-r--r-- 1 xtwang staff 18B Sep 3 14:55 datasets -rw-r--r-- 1 xtwang staff 293B Sep 3 14:55 hg38.chromsizes -rw-r--r-- 1 xtwang staff 29K Sep 3 14:57 tocooler.log
Now, you can visualize BH-FDR and HICCUPS peak annotations on heatmap with peak-plot.
For BH-FDR peaks:
$ peak-plot -O test-BHFDR.png --dpi 250 -p K562-MboI-parts.cool::40000 -I K562-MboI-BHFDR-loops.txt -C 21 -S 29000000 -E 32000000 --correct --siglevel 0.05
The output figure should look like this:
For HICCUPS peaks:
$ peak-plot -O test-HICCUPS.png --dpi 250 -p K562-MboI-parts.cool::40000 -I K562-MboI-HICCUPS-loops.txt -C 21 -S 29000000 -E 32000000 --correct --siglevel 0.1
And the output plot:
The tables below show the performance test of toCooler, pyBHFDR and pyHICCUPS with low (T47D) and high (K562) sequencing data, at low (40K) and high (10K) resolutions.
- Processor: 2.6 GHz Intel Core i7, Memory: 16 GB 2400 MHz DDR4
- Software version: hicpeaks 0.3.0
- At 40Kb resolution, --pw and --ww are set to 1 and 3 respectively; at 10Kb resolution, they are set to 2 and 5 respectively.
- The original Hi-C data is stored in TXT
- Number of proccesses assigned: 1
- Valid contacts: total number of non-zero pixels on intra-chromosomal matrices
- Running time format: hr: min: sec
|Memory Usage||Running time||Memory Usage||Running time||Memory Usage||Running time|
Both pyBHFDR and pyHICCUPS support multiple processes (--nproc). If your computer has sufficient memory, the calculation should end within 30 minutes.
Version 0.3.0 (09/03/2018)
- Remove horizontal and vertical backgrounds for performance
- Support multiple parameters (pw and ww)
- Python 3 support
- Optimize the calculation
- Code refactoring
- Fix bugs when users provide with external .cool files.
Version 0.2.0-r1 (08/26/2018)
- Speeded up the program by dynamically limiting donut width
- Added performance table in README.rst
Version 0.2.0 (08/25/2018)
- Added vertical and horizontal backgrounds
- Added additional filtering based on dbscan clusters and more stringent q value thresholds
- Fixed bugs in storing interchromosomal data
Version 0.1.1 (08/24/2018)
- Lower memory usage and more efficient calculation
Version 0.1.0 (08/22/2018)
- The first release.
- Added toCooler and peak-plot.
- Added multiple process support.
- Implemented core algorithms of BH-FDR and HICCUPS
|||Rao SS, Huntley MH, Durand NC et al. A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping. Cell, 2014, 159(7):1665-80.|
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