Fit-Hi-C

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A Python based command line tool to assign statistical confidence to Hi-C contact maps

View the Project on GitHub ay-lab/fithic

FitHiC and FitHiC2

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Fit-Hi-C (or FitHiC) was initially developed by Ferhat Ay, Timothy Bailey, and William Noble January 19th, 2014. It is currently maintained and updated by Ferhat Ay (ferhatay@lji.org) and Arya Kaul (akaul@lji.org) at the Ay Lab in the La Jolla Institute for Allergy and Immunology.

The current version is named as FitHiC2 (or FitHiC 2.0) due to the addition of many new features compared to FitHiC, like:

1) finding inter-chromosomal significant interactions,

2) applying a merging filter algorithm to filter out putative bystander interactions and keep only the direct CIS chromosomal interactions,

3) Reporting the expected contact count between interacting pairs of bins, along with the raw (observed) contact count, to assess the enrichment of observed from the expected contact count.

Please use the Google Group for discussions/bug reports/analysis questions. Sending an email to fithic@googlegroups.com will also post directly to the Group.

Installation

Fit-Hi-C may be installed through one of three ways.

  1. Bioconda
  2. Github
  3. Pip

Out of all of the following, we recommend installing through bioconda to automatically install all dependencies.

Bioconda Installation

If this is your first time using the conda distribution system, we recommend using Miniconda as your preferred conda distribution system. This is chosen because it is the most lightweight out of all of Anaconda’s distribution system; however, you’re welcome to use any one you would like. More information on each may be found here.

Once your conda distribution platform has been set up, you need to add the bioconda channel to access the bioinformatics recipes hosted there. If you’re unfamiliar with bioconda, I highly recommend you check out the wonderful work they’ve done ( link here!). To set up the bioconda channel, run the following:

conda config --add channels defaults
conda config --add channels conda-forge
conda config --add channels bioconda

Afterwards, simply run:

conda install fithic

This command will automatically install a command-line executable version of Fit-Hi-C along with all of its dependencies. Please run:

fithic -V

and ensure that the version number matches the version number in the Anaconda Cloud badge at the top of this README.

Github Installation

Install git and then run:

git clone https://github.com/ay-lab/fithic.git

You will need the following dependencies installed to run Fit-Hi-C:

This will create a direct clone of Fit-Hi-C in your working directory with the name fithic. You may now run Fit-Hi-Cv.2 by calling the fithic.py file in the fithic/ directory:

python fithic.py --ARGUMENTS

Cloning into the repository does not automatically install the command line version of Fit-Hi-C, and if you desire that functionality follow the other installation instructions.

PyPi Installation

Ensure that you have Python3 successfully installed on your computer. Then run:

pip install fithic

After this is done, run fithic --help to ensure all necessary dependencies have been installed. Some users report that this automatically installs all dependencies, while some say it does not. If fithic does not work properly, install the necessary dependencies using pip. i.e.

pip install DEPENDENCY

Testing

A good part of any software installation is being able to run tests on the correct installation of it.

Bioconda/PyPi Installation

Run the command:

svn export https://github.com/ay-lab/fithic/trunk/fithic/tests/

(If you receive an error, ensure that you have svn installed correctly.) This command will generate a tests folder in your working directory. Going into that and running ./run_tests-cli.sh will automatically run Fit-Hi-C on a variety of data and if everything was installed correctly you should see a final message that Fit-Hi-C executed correctly!

Github Installation

Simply navigate into the repo and run ./fithic/tests/run_tests-git.sh. If everything is working fine, you will see a final message that Fit-Hi-C executed correctly!

Using Fit-Hi-C

Congratulations! If you have gotten to this point, then you have a working, fully installed version of Fit-Hi-C running on your computer. Good on you! But that was the easy part, now comes the difficult question.

How do I correctly use Fit-Hi-C to analyze my desired Hi-C dataset?

Correctly answering this question requires navigating through several basic understandings:

  1. What exactly is Hi-C data?
  2. What does Fit-Hi-C tell me about this Hi-C data?
  3. Why is what Fit-Hi-C tells me important?

If you feel utterly comfortable with the answers to these three questions, then feel free to skip to the next section. If you are unclear about the answer to any of the above, then read on!

What is Hi-C data?

While a beautiful, Latex type-set, easy-to-understand, and comprehensive document is being created, read this !

What does Fit-Hi-C tell me about this Hi-C data?

At the beginning of this README, I stated:

Fit-Hi-C is a tool for assigning statistical confidence estimates to chromosomal contact maps produced by genome architecture assays.

The phrase ‘chromosomal contact maps produced by genome architecture assays’ may be faithfully reduced to ‘Hi-C data.’ Applying that change yields:

Fit-Hi-C is a tool for assigning statistical confidence estimates to Hi-C data.

Much less scary! From the above sentence, the only real phrase that could be misinterpreted is ‘assigning statistical confidence estimates.’ What does that mean? Well to find out you should read the paper Dr. Ay wrote (found here)!

Why is what Fit-Hi-C tells me important?

Fit-Hi-C tells you what contacts are significant. This is incredibly important because not all of the contacts seen in your Hi-C data are truly unexpected interactions. By assigning statistical confidence to each interaction, you will be able to determine which interactions are the most important and consequently, which ones warrant further investigation.

Running Fit-Hi-C

Arguments


Required Arguments

-f, –fragments

The -f argument is used to pass in a full path to what we deem a ‘fragments file,’ Each line will have 5 entries. The second and fifth fields can be any integer as they are not needed in most cases. The first field is the chromosome name or number, the third field is the coordinate of the midpoint of the fragment on that chromosome, the fourth field is the total number of observed mid-range reads (contact counts) that involve the specified fragment. The fields can be separated by space or tab. All possible fragments need to be listed in this file.

One example file would look like below (excluding the header which is not a part of input):

chr extraField fragmentMid marginalizedContactCount mappable? (0/1)
1 0 15000 234 1
1 0 25000 0 0

Note: the file should be gzipped before providing as an input parameter

-i, –interactions

The interactions file contains a list of mid-range contacts between the fragments/windows/meta-fragments listed in the first file above. Each fragment will be represented by its chromosome and midpoint coordinate. Each line will have 5 fields. The first two will represent first fragment, the following two will represent the second and the fifth field will correspond to number of contacts between these two fragments. The fields can be separated by space or tab. Only the fragment pairs with non-zero contact counts are listed in this file.

One example file would look like below (excluding the header which is not a part of input):

chr1 fragmentMid1 chr2 fragmentMid2 contactCount
1 15000 1 35000 23
1 15000 1 55000 12

Note: the file should be gzipped before providing as an input parameter

-o, –outdir

A full path to an output directory of your choice. If it is not already created, it creates if for you.

-r, –resolution

Numerical value indicating resolution of fixed-size dataset being analyzed. If non-fixed size data being studied, set -r 0.


Optional Arguments

-t, –biases

Accepts - a fullpath to a bias file generated by ICE or Knight-Ruiz normalization for Fit-Hi-C with the following format:

chr midpoint bias
1 20000 1.061

Default - None

Description - Bias files help Fit-Hi-C accurately generate statistical significance estimates. If you have it, use it!

Note: the file should be gzipped before providing as an input parameter

-p, –passes

Accepts - Number of spline passes.

Default - 1

Description - Increasing it beyond 2 is unlikely to affect Fit-Hi-C’s output significantly. If you don’t understand what spline fit means then you have not read the paper!

-b, –noOfBins

Accepts - integer representing number of equal occupancy bins you would like Fit-Hi-C to bin your data with

Default - 100

Description - used for spline fitting

-m, –mappabilityThres

Accepts - integer representing the minimum number of hits per locus that has to exist to call it mappable

Default - 1

Description - Increasing it leads to more stringent requirements for treating an interaction as reasonable. Decreasing it leads to less stringent requirements. If you have extremely high resolution data, it may help to bump this up.

-l, –lib

Accepts - String representing prepending information for output files

Default - fithic

Description - Name of the library that is to be analyzed.

-U, –upperbound

Accepts - Integer representing upper bound for the intrachromosomal interactions to be considered in base pairs.

Default - -1 (no limit)

Description - Highly recommended to bound the intrachromosomal interactions being considered.

-L, –lowerbound

Accepts - Integer representing lower bound for the intrachromosomal interactions to be considered in base pairs.

Default - -1 (no limit)

Description - Highly recommended to bound the intrachromosomal interactions being considered.

-v, –visual

Accepts - no argument

Default - None (no plots)

Description - Use if plots of spline fitting are desired. Unfortunately, different matplotlib versions are unstable in different ways. If you’re getting an error, I suggest trying to run Fit-Hi-C without this option and see if that helps.

-x, –chromosome_region

Accepts - ‘interOnly’, ‘intraOnly’, ‘All’

Default - intraOnly

Description -

interOnly is used if you would only like to analyze interchromosomal interactions.

intraOnly is used if youd would only like to analyze intrachromosomal interactions.

All is used if you would like to analyze inter and intrachromosomal interactions.

While you may now be thinking, “Why would I ever not choose ‘All’? More analysis is better!” It is not this simple. Since you are adding significantly more interactions when you analyze interchromosomal and intrachromosomal interactions in tandem, qvalues will be depressed across the board. In addition, few to no datasets are at a high enough resolution to find significanct interchromosomal interactions.

-bL, –biasLowerBound

Accepts - float value of lower bound for bias values

Default - 0.5

Description - bias values below this number will be discarded

-bU, –biasUpperBound

Accepts - float value of upper bound for bias values

Default - 2

Description - bias values above this number will be discarded


Other Arguments

-V, –version

Accepts - No arguments

Default - None

Description - Prints version number. Check to make sure this is the latest version based on version.log file here

-h, –help

Accepts - No arguments

Default - None

Description - prints help message with all options


Output

Each step of Fit-Hi-C, the number of which is user-defined through the -p flag, generates two output files. For step N and library name prefix denoted by ${PREFIX} the two output files will have the following names:

  1. ${PREFIX}.fithic_passN.txt
  2. ${PREFIX}.spline_passN.significances.txt.gz

The first file will report the results of equal occupancy binning in five fields. An example of which is shown below:

avgGenomicDist contactProb stdErr numLocusPairs CCtotal
20077 2.38e-05 2.11e-06 210 19574
20228 1.88e-05 1.44e-06 268 19662
..

The second file will have the exact same lines as in the input file that contains the list of mid-range contacts. This input file had 5 fields as described above. The output from each step will append the following columns to these fields:

1) p-value: p-value of the corresponding interaction, as computed by the binomial distribution model employed in FitHiC.

2) q-value: q-value or FDR obtained by applying Benjamini-Hochberg correction to the p-values.

3) bias1: Bias value of the first interacting fragment.

4) bias2: Bias value of the second interacting fragment.

5) ExpCC: Expected contact count of the current interaction, computed using the raw contact count, spline fit probability of the raw contact count (with respect to the loop distance), and the given bias values. Enrichment of the raw (observed) contact count with respect to the expected contact count is reflected in the q-value.

chr1 fragmentMid1 chr2 fragmentMid2 contactCount p-value q-value bias1 bias2 ExpCC
1 15000 1 35000 23 1.000000e+00 1.000000e+00 1 1.2 22
1 15000 1 55000 12 2.544592e-02 1.202603e-01 1.1 0.9 4
.. .. .. .. ..

Utilities

These utilities are provided as part of Fit-Hi-C (/fithic/utils/) to aid in certain common pre-processing/post-processing steps. They are as follows:

HiCKRy

Regardless of the implementation, we strongly recommend the use of a normalization method in order to have meaningful results for further analysis. The only way for Fit-Hi-C to utilize data from Hi-C normalization is through the bias files. As long as the bias value are scaled to have an average of 1 and high values represent loci with higher overall raw counts, Fit-Hi-C will be able to use them in significance assignment.

HiCKRy is an in-house version of Hi-C contact map normalization using the Knight-Ruiz algorithm for fast matrix balancing. It takes three arguments:

-i,--interactions       Path to the interactions file to generate bias values. Required.
-f, --fragments            Path to the interactions file to generate bias values. Required.
-o, --output            Full path to output the generated bias file to. Required.
-x, --percentOfSparseToRemove     Percent of sparse low contact count loci to remove. The default value is 0.05.

It then outputs a bias file in the format of Fit-Hi-C’s -t input option.

HiCPro2FitHiC

HiC-Pro is a common Hi-C mapping tool used to extract information from the raw reads after the Hi-C assay is run. The following script enables the generation of Fit-Hi-C input directly from HiC-Pro’s output.

It takes the following arguments:

-i MATRIX, --matrix MATRIX     Input matrix file with raw contact frequencies. Required.
-b BED, --bed BED     BED file with bins coordinates. Required.
-s BIAS, --bias BIAS     The bias file provided after IC normalization.
-o OUTPUT, --output OUTPUT     Output path.
-r RESOLUTION, --resolution RESOLUTION     Resolution of the matrix.

The output is the contact maps and fragments file in the format of Fit-Hi-C.

createFitHiCFragments-fixedsize

Generates the fragments file if using a fixed-size resolution with your Hi-C data.

The script takes the following arguments:

--chrLens         Path to a file describing chromosome lengths of the model organism. Required.
--resolution      Resolution of dataset being studied. Required.
--outFile         Full path to the output file desired.

Output is a fragments file in the format of Fit-Hi-C.

createFitHiCFragments-nonfixedsize

A bash script to generate the fragments file if the Hi-C data is RE-digested. Note, order of arguments is critical.

bash createFitHiCFragments-nonfixedsize.sh [outputFile] [RE] [fastaReferenceGenome]
        
[outputFile]               A desired output file path. Required.
[RE]                       Either the name of the restriction enzyme used, or the cutting position using “^”. For example, A^AGCTT for HindIII. Required.
[fastaReferenceGenome]     A reference genome in fasta format. Required.

validPairs2FitHiC-fixedSize

A bash script to generate the contact maps input for Fit-Hi-C from a valid pairs file. Note, order of arguments is critical.

bash validPairs2FitHiC-fixedSize.sh [resolution] [libraryName] [validPairsFile]        

[resolution]         The resolution of the dataset being studied. Required.
[libraryName]        The prefix of the file generated. Required.
[validPairsFile]     A textfile containing the validPairs. Required.

visualize-UCSC.sh

A bash script to convert Fit-Hi-C output into visualization input for UCSC’s Genome Browser in ‘interact’ format.

DESCRIPTION:
bash visualize-UCSC.sh [inputFile] [outputFile] [QvalThresh]
        
         [inputFile]                Input Fit-Hi-C file to visualize 
         [outputFile]               Output file for UCSC to visualize 
         [QvalThresh]               Q-value threshold to filter Fit-Hi-C interactions at 

createFitHiCHTMLout

A bash script to generate an HTML report of the Fit-Hi-C run. Note, works best if Fit-Hi-C was run with --visual option.

bash createFitHiCHTMLout.sh [Library Name] [No. of passes] [Fit-Hi-C output folder]
        
[Library Name]            The library name (-l option) used during Fit-Hi-C’s run
[No. of passes]            The number of spline passes conducted by the Fit-Hi-C run
[Fit-Hi-C output folder]    Path to the output folder for that Fit-Hi-C run (-o option)

createFitHiCContacts-hic.sh

A bash script to create Fit-Hi-C contacts from .hic files.

bash createFitHiCContacts-hic.sh [Juicer's dump command] [chr1] [chr2] [Output file name]
        
[Juicer's dump command]    Full path to the output of Juicer's dump command
[chr1]                     Chromosome 1 of the argument used in Juicer's dump command
[chr2]                     Chromosome 2 of the argument used in Juicer's dump command
[Output file name]          Name of output file

merge-filter.sh

A bash script to merge nearby significant interactions and filter Fit-Hi-C output

bash merge-filter.sh [inputFile] [resolution] [outputDirectory] [fdr]

[inputFile]                Input file of Fit-Hi-C interactions
[resolution]               Resolution used
[outputFile]               Output file to dump output to
[fdr]                      False Discovery rate to use when subsetting interactions
[utilities]                Full path to utilities folder (folder where CombineNearbyInteraction.py is)

merge-filter-parallelized.sh

A bash script to parallelize merge nearby significant interactions and filter Fit-Hi-C output. Note - you will have to modify the actual script contents to assume your cluster config/ organism.

bash merge-filter-parallelized.sh [inputFile] [resolution] [outputDirectory] [fdr]

[inputFile]                Input file of Fit-Hi-C interactions
[resolution]               Resolution used
[outputDirectory]          Directory to dump output to
[fdr]                      False Discovery rate to use when subsetting interactions
[utilities]                Full path to utilities folder (folder where CombineNearbyInteraction.py is)

Citing Fit-Hi-C

If Fit-Hi-C was used in your analysis, please issue the following citations:

1) Arya Kaul, Sourya Bhattacharyya & Ferhat Ay 2020. “Identifying statistically significant chromatin contacts from Hi-C data with FitHiC2.” Nature Protocols. 15:991-1012, 2020. doi: 10.1038/s41596-019-0273-0.

2) Ferhat Ay, Timothy L. Bailey, William S. Noble. 2014. “Statistical confidence estimation for Hi-C data reveals regulatory chromatin contacts.” Genome Research. 24(6):999-1011, 2014. doi: 10.1101/gr.160374.113.

License

Copyright (c), 2012, University of Washington

Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the “Software”), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions:

The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software.

THE SOFTWARE IS PROVIDED “AS IS”, WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.