Find number of off-targets¶
usage: cas_offinder.py [-h] [-j JID] -f INPUT [-n NUM_MISMATCHES] [--add_PAM]
[--remove_first_G] [--PAM_seq PAM_SEQ] [-g GENOME]
[--chr_fa CHR_FA]
optional arguments:
-h, --help show this help message and exit
-j JID, --jid JID enter a job ID, which is used to make a new directory.
Every output will be moved into this folder. (default:
cas_offinder_yli11_2020-03-24)
-f INPUT, --input INPUT
a list of gRNA sequences (default: None)
-n NUM_MISMATCHES, --num_mismatches NUM_MISMATCHES
Number of allowed mis-matches in the gRNA, excluding
PAM sequence (default: 2)
--add_PAM if PAM sequence is not included in your gRNA, please
add this option. (default: False)
--remove_first_G remove first letter G in the input gRNA list (default:
False)
--PAM_seq PAM_SEQ specify the PAM sequence, e.g., NGG. (default: NGG)
Genome Info:
-g GENOME, --genome GENOME
genome version: hg19, hg38, mm9, mm10.(default: hg19)
--chr_fa CHR_FA This will be automatically changed with -g option
(default: /home/yli11/Data/Human/hg19/fasta/chr)
Summary¶
Given a list of gRNA sequences and number of allowed mismatches (excluding PAM), with or without PAM sequences, output number of off-targets (i.e., number of matches up to maximal mismatches) for each gRNA.
Latest updates:
To allow mismatches in the PAM sequence, use
--add_PAM --allow_PAM_mis --PAM_seq NGG, for example:
cas_offinder.py -g hg38 --add_PAM --allow_PAM_mis --PAM_seq NGG -f input2.list -j mis_4_GA -n 4
To allow G-A mismatch in the protospacer sequence, change G to R in the protospacer sequence:
for example ACTGGGAGACACCTCCCAGT becomes ACT``R````R````R``A``R``ACACCTCCCA``R``T
Output example¶
ACTGGGAGACACCTCCCAGTAGG 4058
TGGGAGACACCTCCCAGTAGGGG 4011
CTGGGAGACACCTCCCAGTAGGG 3998
AGGTGTCTGTCGGCCCCTACTGG 3262
GGTGTCTGTCGGCCCCTACTGGG 3208
TCAGGAATTCGAGACCAGCAGGG 3146
GTCTGTCGGCCCCTACTGGGAGG 1687
CAGGAATTCGAGACCAGCAGGGG 919
CTCTCTCCTTGGCCTGCAGATGG 832
GTCAGGAATTCGAGACCAGCAGG 509
Input¶
A list of gRNAs:
ACGACCTTGGCGCCACCACCTGG
TTATCTTTAACACCCCCTGCTGG
AGCTCTCGCACCGCCACTAGAGG
TTACAGGAGAGACCAGATGATGG
CACCGGTGGAATCCAGTAGGGGG
AGTGAGAGGAGGTGCCAGCAGGG
AGCCAGGTGCCGCCCTCCTGAGG
TGGGGGCCTGGGTGTCCACCAGG
GAGCCTTCAGCTACCTCATGTGG
TAGCAGCTGGGAACCAGCAGAGG
Note
if no PAM sequences in the gRNA input, please add --add_PAM option.
Usage¶
Step 0: Load python version 2.7.13.
module load python/2.7.13
Step 1: Run the command
Example command 1: with --add_PAM option.
cas_offinder.py -f gRNA.list2 --add_PAM
2019-07-17 11:09:04,293 - INFO - main - The job id is: cas_offinder_yli11_2019-07-17
2019-07-17 11:09:04,649 - INFO - submit_pipeline_jobs - cas has been submitted; JobID: 83786440
Example command 2: if input gRNA sequences contain PAM, then just run the following command.
cas_offinder.py -f gRNA.list
2019-07-17 11:09:24,777 - WARNING - main - The input job id is not available!
2019-07-17 11:09:24,777 - INFO - main - The new job id is: cas_offinder_yli11_2019-07-17_f0811dd87951
2019-07-17 11:09:24,890 - INFO - submit_pipeline_jobs - cas has been submitted; JobID: 83786441
Note
By default, maximal allowed mismatches is 2. You can control this by -n option.
To find gRNA locations¶
This program can also be helpful to find gRNA coordinates in the genome.
Now, my gRNA list doesn’t have PAM and actually contains G in the beginning of every gRNA. my command will be:
cas_offinder.py -f VPR.gRNA.list -n 0 --add_PAM --remove_first_G
2020-03-24 14:49:45,002 - INFO - main - The job id is: cas_offinder_yli11_2020-03-24
2020-03-24 14:49:45,154 - INFO - submit_pipeline_jobs - cas has been submitted; JobID: 99715775
Output¶
Once the job is finished, you will receive a notification email with the result attached.
In the JobID folder:
match.bed cas-offinder otput bed file (not standard format) showing the matches
match.bed.sorted: sorted standard bed format that are ready to use.
Comments¶
code @ github.