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CCTop is a tool to determine suitable CRISPR/Cas9 target sites in a given query
sequence(s) and predict its potential off-target sites. The online version of
CCTop is available at http://crispr.cos.uni-heidelberg.de/

This is a command line version of CCTop that is designed mainly to allow search
of large volume of sequences and higher flexibility.

If you use this tool for your scientific work, please cite it as:
	Stemmer, M., Thumberger, T., del Sol Keyer, M., Wittbrodt, J. and Mateo, J.L.
	CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool.
	PLOS ONE (2015). doi:10.1371/journal.pone.0124633


REQUIREMENTS

CCTop is implemented in Python and it requires a version 2.7 or above.

In addition we relay on the short read aligner Bowtie 1 to identify the
off-target sites. Bowtie can be downloaded from this site
http://bowtie-bio.sourceforge.net/index.shtml in binary format for the main
platforms.
You need to create an indexed version of the genome sequence of your
target species. This can be done with the tool bowtie-build included in the
Bowtie installation. For that you simply need a fasta file containing the genome
sequence. To get the index you can do something like:
	$ <path-to-bowtie-folder>/bowtie-build -r -f <your-fasta-file> <index-name>

The previous line will create the index files in the current folder.

To handle gene and exon annotations we use the python library bx-python
(https://bitbucket.org/james_taylor/bx-python/). This library is only required
if you want to associate off-target sites with the closest exon/gene, otherwise
you don't need to install it. Notice, however, that in this case all candidate
target sites will be given the same score, because in the current version the
score of the candidate target sites considers only off-target sites with
associated exon/gene.

The exon and gene files contain basically the coordinates of those elements in
bed format (http://genome.ucsc.edu/FAQ/FAQformat#format1), which are the first
three columns of the file. There are two more columns with the ID and name of
the corresponding gene and a sixth empty column to comply with the format
accepted by the library. You can generate easily such kind of files for you
target organism using Ensembl Biomart (http://www.ensembl.org/biomart).

In case of difficulties with these files contact us and we can provide you the
ones you need or help you to generate you own.


INSTALLATION

Simply download the two .py files (CCTop.py and bedInterval.py) to a folder of
your choice and follow the instructions that you can find in the respective web
sites to install Bowtie 1 and bx-python.


USAGE

You can run CCTop with the -h flag to get a detailed list of the available
parameters. For instance:
	$ python <download-folder>/CCTop.py -h<Enter>

At minimum it is necessary to specify the input (multi)fasta file (--input) and
the index file (--index). In this case CCTop assumes that the Bowtie executable
can be found in the current folder, there are not gene and exon file to use and
the rest of parameters will take default values. Notice that the index file to
specify refers to the name of the index you specified for bowtie-build together
with the path, if necessary. A command for a typical run will look something
like this:
	$ python <download-folder>/CCTop.py --input <query.fasta> --index <path/index-name> --bowtie <path-to-bowtie> --output <output-folder> <Enter>

The result of the run will be three files for each sequence in the input query
file. These files will have extension .fasta, .bed and .xls, containing,
respectively, the sequence of the target sites, their coordinates and their
detailed information as in the online version of CCTop. The name of the output
file(s) will be taken from the name of the sequences in the input fasta file.