-m or –mode {str}: This parameter allows for the specification of the type of analysis: “CRISPResso” and “CRISPResso2” is allowed in the latest version
-a or –amplicon_seq {str}: This parameter allows the user to enter the amplicon sequence used for the CRISPResso. The length should be >105bp due to the setting for the other parameter.
-g or –guide_seq {str}: This parameter allows for the specification of the sgRNA sequence used for the CRISPResso. The length of the sequence should be 20nt without PAM. The MaChIAto convention is to depict the expected cleavage position using the value of the parameter three nt 3’ from the end of the guide.”, required=True)
-cf or –crispreeso_file {str}: This parameter allows for the specification of the “Alleles_frequency_table.txt” from CRISPResso. When this parameter is used, “CRISPResso” should be entered as -m parameter.’, (default: “./Alleles_frequency_table.txt”) (optional)
-ccf or –crispreeso2_file {str}: This parameter allows for the specification of the “Alleles_frequency_table.zip” from CRISPResso2. When this parameter is used, “CRISPResso2” should be entered as -m parameter.’, (default: “./Alleles_frequency_table.zip”) (optional)
-d or –donor_seq {str}:This parameter allows for the specification of the expected HDR amplicon used for the CRISPResso. The length of sequence should be >12bp in knock-out/knock-in analysis. In knock-out analysis, this parameter should not be entered, and then the value will be given fake parameter (“TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT”), and some internal settings are changed for knock-out analysis. However, the fake parameter will be poly-C|G|A if amplicon sequence contains poly-T sequence.’, (default: “”) (optional)
-e or –expected_ki_amplicon_seq {str}: This parameter allows for the specification of the expected knock-in amplicon sequence used for the CRISPResso after HDR. The length of the sequence should be >12bp. In knock-out analysis, this parameter should not be entered, and then the value will be given fake parameter including fake donor sequence, and some internal settings are changed for knock-out analysis.’, (default: “”) (optional)
-o or –output_folder {str}: This parameter allows for the specification of the output directory to use for the analysis (default: current directory) (default: “./“) (optional)
-lh or –length_left_homologyarm {int}: This parameter allows for the specification of the length of 5’ homology arm (default: 20). The length of the sequence should be >17bp, and the flanking sequence with the homology arm needs the length of >24bp in the expected amplicon sequence. In the prime editing analysis, the 5’ homology arm is considered as the RT template (default: 20) (optional)
-rh or –length_right_homologyarm {int}: This parameter allows for the specification of the length of 3’ homology arm (default: 20). The length of the sequence should be >17bp, and the flanking sequence with the homology arm needs the length of >24bp in the expected amplicon sequence. In the prime editing analysis, the 3’ homology arm is considered as the prime binding site (default: 20) (optional)
-cn or –location_comp_nick {int}: This parameter allows for the specification of the complementary strand nick location [3prime direction is +] (default: 90). This parameter is used in the prime editing and should be over the homology arm’s length to which the nickase is adjacent’ (default: 90) (optional)
-n or –name : This parameter allows for the specification of the name, which will be included output directory (default: “untitled-X”). If MaChIAto Analyzer and MaChIAto Reviewer will be used in the following analysis, the value should be “{target_name}-{sample label }” (e.g., DBF4B-C) and underbar “_” should not be used’, (default: “untitled-X”) (optional)
–primeediting_analysis : Re-classify the data as prime editing analysis. This option forces the setting to change for prime editing analysis.’ (optional)
Advanced parameter (for developers)
–force_knockout_analysis : Usually, MaChIAto re-classify the data as knock-in analysis. This option forces the setting to change for knock-out analysis. Under this mode, the length of indicator on the knock-in donor is the maximum value, and the threshold value for alignment of the knock-in sequence is 1.0. If this parameter is not entered, MaChIAto can automatically set up this mode by finding some characteristics of the knock-out sample. For example, MaChIAto checks that there is no donor sequence or expected knock-in sequence as input, and there are less three kinds HDR variants among input data. (optional)
–skip_optimization : Usually, MaChIAto runs Bayesian optimization for finding the optimized setting. This option allows MaChIAto to skip the process of optimization. The option is made for debugging. So, the option should not be used in the usual analysis. However, if the optimization process disturbs an accurate analysis, this option might be useful. (optional)
–copy_optimization : Usually, MaChIAto runs Bayesian optimization for finding the optimized setting. This option allows MaChIAto to use the provided optimization data instead of the optimization. If you analyze NGS data with the setting of previously analyzed data, this option is useful. Especially in substitution editing, we can recommend applying the option to the negative/positive control sample. However, you should understand that a classification error might frequently occur. (optional)
–provided_optimization_file : This parameter allows for the specification of the “MaChIAto_optimized_param.csv” from the analyzed MaChIAto folder. When this parameter is used, –copy_optimization parameter is required.’, (default: “./MaChIAto_optimized_param.csv”) (optional)
NOTE:
You can skip the optimization process using the “–skip_optimization” flag. However, we recommend you not to use the option unless you check the command and perform debugging. For example, suppose executing MaChIAto Classifier with poor optimization cause missing the many reads that include expected edits. Moreover, if the indicator is accidentally located in a spontaneous mutation point, The Bit-by-Bit Alignment might misrecognize the editing read. The optimization can avoid the problem. Thus, it would be best if you turned on the optimization for finding the bona fide editing reads.
Template command
Case of knock-out
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python MaChIAto/MaChIAto.py \ -m CRISPResso2 \ -ccf (Alleles_frequency_table.zip of the CRISPResso2 output) \ -o (output directory) \ -a (wt amplicon sequence) \ -g (protospaser sequence) \ -n (sample name)-(label name);
Case of Prime Editing (Substitution/Deletion editing)
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python MaChIAto/MaChIAto.py \ -m CRISPResso2 \ -ccf (Alleles_frequency_table.zip of the CRISPResso2 output) \ -o (output directory) \ -a (wt amplicon sequence) \ -g (protospaser sequence) \ -e (expected editing amplicon seqeunce) \ -lh (length of prime binding site) \ -rh (length of RT template) \ -cn (distance between two nick sites) \ --primeediting_analysis \ -n (sample name)-(label name);
Case of Prime Editing (Insertion editing)
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python MaChIAto/MaChIAto.py \ -m CRISPResso2 \ -ccf (Alleles_frequency_table.zip of the CRISPResso2 output) \ -o (output directory) \ -a (wt amplicon sequence) \ -g (protospaser sequence) \ -e (expected editing amplicon seqeunce) \ -d (donor insert sequence) \ -lh (length of prime binding site) \ -rh (length of RT template) \ -cn (distance between two nick sites) \ --primeediting_analysis \ -n (sample name)-(label name);
If you want to use the result of CRISPResso version1, the -ccf should be replased into -cf. Example for CRISPResso version1
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python MaChIAto/MaChIAto.py \ -m CRISPResso \ -cf (Alleles_frequency_table.txt of the CRISPResso output) \ -o (output directory) \ -a (wt amplicon sequence) \ -g (protospaser sequence) \ -n (sample name)-(label name);
*The sample name and label name can be arbitrary. If you analyze the multiple experiment (e.g. knock-out and knock-in), the name should be different from others.
