Table 1.

All command line parameters for the miRMut scripts

Parameter	Function	Scripts that accepts the parameter
--from_step, -s	Accepts a number value from 1 to 6. Starts analysis from chosen step (inclusive), where: 1 – Filtering miRNA gene mutations 2 – Processing mutation list 3 – Adding miRNA-specific information to specific mutations 4 – Calculating mutation weights and HGVS nomenclature 5 – Generating summaries 6 – Generating visualization of mutations Note that previous steps need to be run before as each step depends on output files from earlier steps. Note that --from_step parameter is overwritten when --csv_file parameter is used if defined --from_step value is lower than 3.	run_mirnaome_analysis.py
--end_step, -es	Accepts a number value from 1 to 6.  Stops the analysis on the chosen step (inclusive). For numbers description see --from_step parameter.	run_mirnaome_analysis.py
--include_merger, -m	Accepts values 0 (disabled) or 1 (enabled). It must be enabled if analysed VCF/VCF.GZ files were created with multiple algorithms (e.g., MuSE, MuTect2, etc.). If the merger of algorithms is enabled, mutations are grouped by position, individual ID of patient, and mutation type (reference and alternative allele), and if the same mutation is found in files generated by different algorithms, it is treated as one mutation. This transformation prevents counting the same mutation multiple times. If the merger of algorithms is enabled, the “indiv_name” has to be defined in the VCF/VCF.GZ files.	run_mirnaome_analysis.py, merge_algorithms.py
--include_filtering, -f	Accepts values 0 (disabled) or 1 (enabled). If filtering is enabled, for each “results_{file type}.csv” file, “results_{file type}_eval.csv” is created with an additional column “eval”, which contains information if the mutation passes filtering as defined below. If filtering is enabled, the following criteria are taken into account (only if available in VCF/VCF.GZ files): •SSC > 30 •BQ of an alternative allele in tumor sample > 20 •QSS of an alternative allele in tumor samples divided by alternative allele count in tumor samples > 20 •At least two mutation-supporting reads in a tumor sample (if no mutation-supporting read was detected in the corresponding normal sample) OR at least 5 × higher frequency of mutation-supporting reads in the tumor sample than in the corresponding normal sample Please note that some of the filtering criteria are strictly algorithm-related and are not relevant in other projects.	run_mirnaome_analysis.py, merge_algorithms.py
--csv_file, -c	Accepts path to a CSV file. The parameter is used when the miRMut is to be run on a CSV file instead of VCF/VCF.GZ files. The CSV file needs to be formatted as defined in adding miRNA-specific information to specific mutations section. When used with run_mirnaome_analysis.py script, the input folder argument will not be used (but needs to be defined), and the script will automatically start from step 3 (unless a later step is defined using --from_step parameter), skipping the first two steps.	run_mirnaome_analysis.py, add_mirna_info.py
--pass_arg, -p	Accepts values 0 (disabled) or 1 (enabled). When PASS filtering is enabled using --pass_arg parameter only mutations with PASS value in the FILTER column from VCF/VCF.GZ files are included in the analysis. Although there is this option, it is recommended to do VCF/VCF.GZ filtering prior to the miRMut usage.	run_mirnaome_analysis.py, extract_results_for_mirnaome.py
--weight_filter, -w	Accepts values 1, 1.5 and 2. The default value is set to 1. It filters out only mutations with selected weight or higher. It is done on the 4th step of the script and affects files with hgvs nomenclature, summaries and figures.	run_mirnaome_analysis.py, add_weghts.py