Run RawTools for quality control for Windows
To run the quality control analysis with RawTools, we will use the same raw data file that we used previously for the parsing and quantification analysis. For this walkthrough we are using Command prompt (type command prompt in the Windows search to locate the program).
First navigate to the RawTools directory. We are still using the rawTools folder location on our system.
>D:
>cd rawTools
Load up the help file for RawTools QC:
>RawTools qc --help
As you can see, there are many parameters for the QC analysis with RawTools. Let's first do a basic QC analysis with no database search.
>RawTools qc -d D:\rawTools\ -q D:\rawTools\qc_output\
In the command above, the -d flag is telling RawTools the directory to look for raw files. Every file in the specified directory will be processed to a single output. The -q flag is telling RawTools where to write the output data. This folder does not need to exist prior to the analysis. The Command prompt output should appear similar to below:
Within the qc_output folder, a QC.xml tracking folder and a QcDataTable.csv that contains the summarized outputs will both be made. For further details on how to interpret the QC output, please visit the descriptive page.
To perform a QC analysis with a database search, we will need to use a few more parameters. You will also need a fasta sequence database. We will be using a human database from UniProt. Input the command below:
>RawTools qc -d D:\rawTools\ -q D:\rawTools\qc_output_wSearch\ -i --db D:\rawTools\uniprot_human.fasta --fmods 57.0214@C -xmod 15.9949@M -N 1000
This command may need modification depending on your system. Specifically, the -p and -I flags are trying to locate the python and identipy bin files. If you encounter issues tracking these files down, please make a post on the Issues page of this GitHub and we will help you.
In this command, the -i flag is telling RawTools to do an IdentiPy search. The --db input is giving the path to the fasta database to use in the IdentiPy search. This should just be a normal forward database, with no decoy sequences. The --fmods is giving the fixed modification to the IdentiPy search. The --xmod, --nmod, and --kmod are all variable modification inputs. The --xmod flag is for individual variable modifications, like oxidation or any others. For isobaric tagging, use the --nmod and --kmod tags. The Command prompt output should appear similar to below:
As above, for further details on how to interpret the QC output, please visit the descriptive page.