hc1023 / casc

CASC Ain't Simply CRT

Welcome to CASC, an all-in-one CRISPR detection and validation tool designed for CRISPR discovery in metagenomic or genomic reads or contigs.

CASC, short for "CASC Ain't Simply CRT", is a tool which utilizes a modified version of the CRISPR Recognition Tool (CRT) to call putative CRISPR spacers. CASC then validates these spacers by searching against a database of Cas proteins and CRISPR repeats to get rid of false-positives.

If your input FASTA file contains at least one CRISPR spacer, CASC will output seven files:

• casc.log: A log with info about your last casc run
• file.bonafide.spacers.fasta: FASTA file containing 'bona fide' or 'valid' CRISPR spacers
• file.non-bonafide.spacers.fasta: FASTA file containing 'non-bona fide' or 'non-valid' CRISPR spacers (i.e. those appearing to be false-positive from CRT)
• file.bonafide.repeats.fasta: FASTA file containing 'bona fide' or 'valid' CRISPR repeats
• file.non-bonafide.repeats.fasta: FASTA file containing 'non-bona fide' or 'non-valid' CRISPR repeats
• file.report.md: Markdown file with some summary statistics on your run
• file.results.txt: Tab-delimmited breakdown of results

1. Downloading CASC

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NOTE: CASC was written on Mac OS, and therefore will only work on UNIX-based operating systems (e.g. Mac OS, Linux).

To download, simply clone the CASC repository from GitHub. From the commandline type:

$ git clone https://github.com/dnasko/CASC.git

And CASC will be cloned to your working directory.

2. Installing CASC and its Dependencies

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Installing CASC system-wide

If you have sudo acces you can install CASC easily:

perl Makefile.PL
make
make test
sudo make install

Installing CASC locally

If you would like to install CASC without sudo:

perl Makefile.PL PREFIX=/Path/to/where/to/install
make
make test
make install

By installing this way you will need to update your PATH and @INC. This is done by adding the following to your ~/.profile or ~/.bash_profile:

PATH=$PATH:/Path/to/where/to/install/bin
export PATH
export PERL5LIB=/Path/to/where/to/install/lib/perl5/site_perl

Of course, you need to replace "/Path/to/where/to/install" with where you actually installed it ;)

CASC HAS ONLY ONE EXTERNAL DEPENDENCY, and it's BLAST. You will need to have a local copy of BLAST installed on your machine, and have its location in your PATH. ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/

Assuming you have Perl installed on your machine (and BLAST) you need only to execute the following command to display CASC's help / usage:

casc --help

3. Using CASC to Find CRISPRs

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Say you have a recently sequenced genome, or an assembled metagenome saved in a file TestSeqs.nuc.fasta and you would like to find CRISPR spacers within this genome or library. You would execute the following:

casc -i TestSeqs.nuc.fasta -o outdir

CASC does come with two optional arguments, allowing you to choose how many CPUs you would like to use, and whether or not you would like to be conservative or liberal with CRISPR call. By default you will only use 1 CPU, and call CRISPRs liberally. In this example we are saving to a new folder on our home directory, using 4 CPUs, and calling CRISPRs conservatively:

casc -i /Path/to/TestSeqs.nuc.fasta -o NewOutput -n 4 --conservative

The CASC "*.results.txt" file

The results.txt file provides useful results for each array found. The fields are pretty self explanatory. Well, all but one. The final field, "code" is a bit cryptic. It is a single digit integer (between 0-7) that codifies certain results, shown below:

Code	(Binary)	Cas Protein Hit	Matches Known Repeat	Proper Statistics
0	000	no	no	no
1	001	no	no	Yes
2	010	no	Yes	no
3	011	no	Yes	Yes
4	100	Yes	no	no
5	101	Yes	no	Yes
6	110	Yes	Yes	no
7	111	Yes	Yes	Yes

4. Version History

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CASC is routinely updated in an effort to assure that you are validating CRISPRs with the most up-to-date versions of UniRef and CRISPR DB:

Version	Date	Notes
2.6	14Feb2017	Now uses MakeMaker for installation; Various improvements to results reporting
2.5	29Sep2015	UniRef DB updated to 29Sep2015; various bug fixes
2.4	07Oct2014	CRISPR DB updated to 07Oct2014; UniRef DB updated to 07Oct2014; added -silent argument
2.3	17Feb2014	CRISPR DB updated to 17Feb2014; UniRef DB updated to 17Feb2014; added -v argument
2.2	02Jun2013	Improved report formatting and added array coordinates to each array
2.1	09Apr2013	Various bug fixs, improved report format
2.0	22Mar2013	Improved multithreading function
1.2	17Mar2013	Linux compatibility improved, conservative option added
1.1	16Mar2013	CRISPR DB updated to 29Jan2013; UniRef DB updated to 16Mar2013
1.0	19Dec2012	Initial release

5. Credits

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CASC would not be possible without the help of others who have written some very nice software and performed exceptional research. CASC uses a modified version of the CRISPR Recognition Tool (CRT). From there, CASC will then validate these putative spacers by using a BLAST homology search against the CRISPR Finder Repeat Database. Lastly a final BLASTX is performed against all UniRef100P clusters which represent CRISPR-associated proteins (Cas). All of these citations are included below:

• Bland C, Ramsey TL, Sabree F, Lowe M, Brown K, Kyrpides NC, Hugenholtz P: CRISPR Recognition Tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics. 2007 Jun 18;8(1):209.
• The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics. 2007 May 23;8(1):172.
• S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403–410.
• Suzek,B.E., Huang,H., McGarvey,P., Mazumder,R. and Wu,C.H. (2007) UniRef: comprehensive and non-Redundant UniProt reference clusters. Bioinformatics, 23, 1282Ð1288.

Enjoy!