CRISPREDetect is a perl program developed and tested in Fedora 21 Linux operating system. CRISPRDetect.pl should run under any unix based operating system that has a working 'perl' executable [comes with default installations under all *nix based operating systems]. If all the 3rd party dependencies are installed and available in user/system $PATH CRISPRDetect should run without any issues.
The cd-hit-est program installation in Mac operating system were reported to have issues. If you face issues installing cd-hit-est, please refer to the link: weizhongli/cdhit#24
Please make sure that the following 3rd party tools are installed in your system. The only CPAN perl package 'Parallel' is provided in the 'lib' folder and should work. However, if it doesn't then either execute "cpan Parallel::ForkManager" to install it or execute the following commands after making sure you have its dependencies (POSIX, Storable, File::Spec, File::Temp, File::Path 2.00 and Test::More 0.81_01) installed in your system
wget http://search.cpan.org/CPAN/authors/id/Y/YA/YANICK/Parallel-ForkManager-1.19.tar.gz
tar xvzf Parallel-ForkManager-1.19.tar.gz
cd Parallel-ForkManager-1.19
perl Makefile.PL && make test && make install
The following dependencies are needed by CRISPRDetect.
clustalw Download from ftp://ftp.ebi.ac.uk/pub/software/clustalw2/2.1/
water Comes with EMBOSS:6.3.1+ tools : Download from ftp://emboss.open-bio.org/pub/EMBOSS/EMBOSS-6.6.0.tar.gz
seqret Comes with EMBOSS:6.3.1+ tools : Download from ftp://emboss.open-bio.org/pub/EMBOSS/EMBOSS-6.6.0.tar.gz
RNAfold Comes with Vienna RNA package : Download the correct version specific for your operating system from http://www.tbi.univie.ac.at/RNA/#download
cd-hit-est Comes with cdhit package : Download from http://weizhongli-lab.org/cd-hit/download.php
blastn Comes with ncbi-blast+ package : Download from ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/
CRISPRDetect requires clustalw2 to be available in the $PATH as clustalw. You can do that by "cp /PATH_TO_CLUSTALW2/clustalw2 /bin/clustalw" or by creating a symbolic link "ln -s /PATH_TO_CLUSTALW2/clustalw2 /bin/clustalw" where PATH_TO_CLUSTALW2 is the full path of the directory where the executable clustalw2 is present.
Once all the dependencies are installed, please check that they are successfully installed and available in the user/system PATH by typing the following:
clustalw -help
water -help
seqret -help
RNAfold -help
cd-hit-est -help
blastn -help
perl CRISPRDetect.pl -g NZ_CP006019.gbk -o NZ_CP006019_CRISPRDetect.txt > NC_003106_CRISPRDetect.log
The above command runs CRISPRDetect with default paramaeter on a complete gbk file (file containing both annotation and sequence) that has cas1 or cas2 annotated.
perl CRISPRDetect.pl -f test_multifasta.fa -o test_CRISPRDetect -check_direction 0 -array_quality_score_cutoff 3 -T 0 > test.log
The above command runs CRISPRDetect with a lower score cutoff on a fasta file, cutoff 3 rather than 4, as cas1 and cas2 are not annotated and would score +1. Appropriate for contigs/fasta. -T 0, use all processors rather than the default of 4. Does not check direction (not recommended) ]
-f/-g FASTA/Genbank Specify a FASTA formatted [e.g. -f test.fa] or Genbank formatted file [e.g. -g NZ_CP006019.gbk] containing the sequence.
Note: the default cutoff of 4 is appropriate for genbank files that have cas1 or cas2 annoated, 3 is more appropriate for fa.
-o TEXT Specify a text file that will contain the output [e.g. -o NC_003106_CRISPRDetect]
Note: CRISPRDetect will provide two additional output files - one containing the filtered out arrays (e.g. NC_003106_CRISPRDetect.fp)
and a gff annotation file (e.g. NC_003106_CRISPRDetect.gff)
-h/-help HELP shows this help text
-q/-quiet 0 or 1 Switch off/on step by step progress reporting [default 0]
-T Threads Specify number of parallel processes CRISPRDetect should use [default 4; specify '-T 0' to use all processors]
-tmp_dir tmp/ This is the default directory where temporary files generated by CRISPRDetect and its dependencies will be stored.
-word_length 11 This is the default word length CRISPRDetect uses to find the putative CRISPRs. Any positive integer >=6 can be used.
-minimum_word_repeatation 3 By default CRISPRDetect uses 3 repeating identical words to find putative CRISPRs. To find CRISPRs with 2 repeats, use -minimum_word_repeatation 2
-max_gap_between_crisprs 125 By default the maximum gap is set tp 125 nucleotides between the repeating identical seed words.
-repeat_length_cutoff 17 After the intial processing, putative CRISPRs with repeat lengths less than this value will be rejected.
-minimum_repeat_length 23 Minimum length of repeats
-minimum_no_of_repeats 3 Predicted CRISPRs with number of repeats less than this value will be excluded. To include CRISPRs with only 2 repeats, use -minimum_no_of_repeats 2
-array_quality_score_cutoff 4 Predicted CRISPRs with score less than this value will be excluded from the output file.
The CRISPRs with score >=0 and less than the specied value will be moved to the output.fp file [output refers to user given output filename]. Cutoff of 3 is more appropriate for fasta files.
-left_flank_length 500 This is the default length of the 5' (upstream) region of the CRISPRs.
-right_flank_length 500 This is the default length of the 3' (downstream) region of the CRISPRs.
To test different methods as specified in the literature, open the CRISPRDetect.pl program with any text editor [e.g. gedit in RHEL/Fedora/CentOS, or vi in any *nix OS, or notepad in Windows OS] and change the parameters in the top most section of the script. To toggle individual methods, locate the '$check_' prefix and change the value to 1 (i.e. the method will be applied) or 0 (i.e. the method will not be applied).
Examples:
Direction specific options:
--------------------------
$check_direction=0; [ Default is 1, making it 0 will turn the method off.]
To change the parameter(s) of a particular method (such as check_array_degeneracy) change the nested variables under that particular method.
$check_array_degeneracy=1;
$array_degeneracy_score=0.41; [ Default: PPV (0.91) - 0.50 ]
$permitted_mutation_per_array=0; [ Default 0 ]
Changing to '$permitted_mutation_per_array=2;' will instruct the program to allow maximum 2 bases as permitted mutations per CRISPR array.
The 'tmp' folder in the CRISPRDetect installation directory should have read and write permissions. An easy way to do that is by issuing the command 'chmod -R 755 . && chmod 777 tmp' from the CRISPRDetect installation directory. If you use CRISPRDetect, then please cite:
Biswas, A., Staals, R. H., Morales, S. E., Fineran, P. C. & Brown, C. M. CRISPRDetect: a flexible algorithm to define CRISPR arrays. BMC Genomics 17, 356 (2016).
For version updates and bug fixes refer to https://github.com/ambarishbiswas/CRISPRDetect_2.2 http://bioanalysis.otago.ac.nz/CRISPRDetect
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