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Welcome to hybkit, a toolkit for analysis of “.hyb” (hyb-file) format chimeric (hybrid) RNA sequence data defined with the Hyb software package by Travis et al. (Methods 2014). This genomic data-type is generated from RNA proximitiy-ligation and ribonomics techniques such as Crosslinking, Ligation, and Sequencing of Hybrids (CLASH; Helwak et al. (Cell 2013)) and Quick CLASH (qCLASH; Gay et al. (J. Virol. 2018)).
This software is available via Github, at http://www.github.com/RenneLab/hybkit .
Full project documentation is available at this documentation.
Project components:
  1. hybkit toolkit of command-line utilities for manipulating, analyzing, and plotting hyb-format data.

  2. The hybkit python API, an extendable documented codebase for creation of custom analyses of hyb-format data.

  3. Example analysis pipelines for analysis of publicly available qCLASH hybrid sequence data implemented either with toolkit scripts or the hybkit Python API.

Hybkit Toolkit:

The hybkit toolkit includes several command-line utilities for manipulation of “.hyb” format data:




Parse a hyb file and check for errors


Evaluate hyb records to identify segment types and miRNAs


Filter a hyb file to a specific subset of sequences


Perform a type, miRNA, summary, or target analysis on a hyb file


Filter a fold file using an exclusion table created by hyb_filter


Perform a fold analysis on a hyb and a RNA secondary structure (fold) file in “.vienna” or “.ct” format on a hyb file

These scripts are used on the command line with hyb files. For example, to filter a hyb file to contain only hybrids with a sequence identifier containing the string “kshv”


$ hyb_filter -i my_hyb_file.hyb --filter any_seg_contains kshv

Further detail on the usage of each script is provided in the hybkit Toolkit section of this documentation.

Hybkit API:

Hybkit provides a Python3 module with a documented API for interacting with records in hyb files. This capability was inspired by the BioPython Project. The primary utility is provided by classes for hyb records (HybRecord), classes for fold records (FoldRecord, DynamicFoldRecord), and file-iterator classes (HybFile, ViennaFile, CTFile). Record attributes can be analyzed, set, and evaluated using included class methods.

For example, a workflow to print the identifiers of only sequences within a “.hyb” file that contain a miRNA can be performed as such:

#!/usr/bin/env python3
import hybkit
in_file = '/path/to/my_hyb_file.hyb'

# Open a hyb file as a HybFile Object:
with hybkit.HybFile.open(in_file, 'r') as hyb_file:

    # Return each line in a hyb file as a HybRecord object
    for hyb_record in hyb_file:

        # Analyze each record to assign segment types

        # If the record contains a long noncoding RNA type, print the record identifier.
        if hyb_record.has_prop('any_seg_type_contains', 'lncRNA')

Further documentation on the hybkit API can be found in the hybkit API section of this documentation.


Hybkit provides several example pipelines for analysis of “hyb” data using the utilities provided in the toolkit. These include:



Summary Analysis

Quantify the sequence and miRNA types in a hyb file

Target Analysis

Analyze targets of a set of miRNAs from a single experiment

Grouped Target Analysis

Analyze and plot targets of a set of miRNAs from pooled experimental replicates

Fold Analysis

Analyze and plot predicted miRNA folding patterns in miRNA-containing hybrids

These pipelines provide analysis results in both tabular and graph form. As an illustration, the example summary analysis includes the return of the contained hybrid sequence types as both a csv table and as a pie chart:

Further detail on each provided pipeline can be found in the Example Pipelines section of this documentation.


The recommended installation method is via hybkit’s PyPI Package Index using python3 pip, which will automatically handle version control and dependency installation:

$ pip install hybkit

Acquisition of the package can also be performed by cloning the project’s Github repository:

$ git clone git://github.com/RenneLab/hybkit

Or by downloading the zipped package:

$ curl -OL https://github.com/dstrib/hybkit/archive/master.zip
$ unzip master.zip

Followed by installation using python’s setuptools:

$ python setup.py install

Further documentation on hybkit usage can be found in this documentation.

hybkit is a free, sharable, open-source project.
All source code and executable scripts contained within this package are considered part of the “hybkit” project and are distributed without any warranty or implied warranty under the GNU General Public License v3.0 or any later version, described in the “LICENSE” file.