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Using the Command Line

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Alexander-Sol edited this page Mar 19, 2025 · 19 revisions

A command line version of FlashLFQ can be obtained by downloading the "FlashLFQ.zip" asset from any FlashLFQ release

Use the --help argument to get a list of arguments. For example, on Windows:

CMD.exe --help

At minimum, the --idt (the identification file) and --rep (the spectra file repository) arguments must be specified.

If you want to normalize results or perform Bayesian statistical analysis using the command-line, you must create an experimental design TSV file and place it in the directory with your spectra files (see Experimental Design wiki page).

For boolean (true/false) parameters, an absent flag means "false" and a present flag means "true".

For example, the chg parameter will be set to true here:

CMD.exe --idt "C:\MyFolder\msms.txt" --rep "C:\MyFolder" --ppm 5 --chg

The chg parameter will be set to false here:

CMD.exe --idt "C:\MyFolder\msms.txt" --rep "C:\MyFolder" --ppm 5

false is the default state for all of FlashLFQ's Boolean parameters.

Processing Thermo .raw files

If you would like to process Thermo .raw data files directly, you must agree to the Thermo license. This is accomplished by creating a text file with the name "LicenseAgreements.toml" with the text below. This file should be placed in the same directory as the executable program.

HasAcceptedThermoLicence = true

Windows

To use the FlashLFQ standalone command-line version, run the "CMD.exe" program with command-line arguments.

Preferably, when specifying a filepath, use the absolute file path inside of quotes.

Example:

CMD.exe --idt "C:\MyFolder\msms.txt" --rep "C:\MyFolder" --ppm 5 --chg

Linux

FlashLFQ is best run in Linux via .NET Core. You can also use Mono, though historically there have been RAM issues. You can run the .NET Core version of FlashLFQ with the "dotnet CMD.dll" command. You will likely not be able to open Thermo .raw files in Linux; you should convert them to .mzML before quantifying with FlashLFQ.

Example:

dotnet CMD.dll --idt "/home/myfolder/msms.txt" --rep "/home/myfolder" --ppm 5 --chg

Command-Line Arguments

Required Arguments

  • --idt (string): Path to the identification file.
  • --rep (string): Directory containing spectral data files.

Optional Arguments

Input and Output

  • --pep (string, optional): Path to the all-peptide file.
  • --out (string, optional): Output directory.

Processing Settings

  • --nor (bool, default: false): Normalize intensity results.
  • --ppm (double, default: 10): PPM tolerance.
  • --iso (double, default: 5): Isotopic distribution tolerance in ppm.
  • --int (bool, default: false): Integrate peak areas (not recommended).
  • --nis (int, default: 2): Number of isotopes required to be observed.
  • --chg (bool, default: false): Use only precursor charge state.
  • --thr (int, default: -1): Number of CPU threads to use. Default argument (-1) causes FlashLFQ to use one less than the maximum number of threads

Match Between Runs (MBR) Settings

  • --mbr (bool, default: true): Enable Match-Between-Runs (MBR) / Peptide-Identity-Propagation (PIP).
  • --mrt (double, default: 1.5): Maximum MBR window in minutes.
  • --rmc (bool, default: false): Require MS/MS ID in condition.
  • --pipfdr (double, optional): FDR cutoff for MBR/PIP.
  • --usepepq (bool, default: false): Use PEP Q Value to determine peptides to quantify. Only relevant for MetaMorpheus results.

Bayesian Protein Quantification Settings

  • --bay (bool, default: false): Enable Bayesian protein fold-change analysis.
  • --ctr (string, optional): Control condition for Bayesian analysis.
  • --fcc (double, default: 0.1): Fold-change cutoff for Bayesian analysis.
  • --mcm (int, default: 3000): Number of MCMC iterations.
  • --bur (int, default: 1000): Number of MCMC burn-in iterations.
  • --sha (bool, default: false): Use shared peptides for protein quantification.
  • --rns (int, optional): Random seed for Bayesian analysis.

License and File System Settings

  • --sil (bool, default: false): Silent mode.
  • --rea (bool, default: false): Read-only mode (prevents writing to FlashLFQ folder).
  • --pth (bool, default: false): Print Thermo's RawFileReader license.
  • --ath (bool, default: false): Accept Thermo's RawFileReader license.

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