FragPipe for Linux (AAS Project)

FragPipe, developed by the Nesvizhskii Lab, is a computational platform designed for analyzing mass spectrometry-based proteomics data. Its documentation is thorough and found here, but this repository is dedicated to describing how to employ FragPipe in Linux specifically for the Amino Acid Substitution Project.

This implementation is optimized for using FragPipe v24.0 and its dependencies to search multiplexed (TMT-labelled) proteomics data with custom FASTA files containing substituted sequences.

Dependencies

1. Results of MaxQuant Dependent Peptide search
2. Results of SAAP_Detection and Validation1 Python scripts from the Decode Pipeline
3. FragPipe v24.0 and its dependencies
4. All scripts in this repository

Recommended directory formats on HPC

/home/maropakis.a/scripts/
└── Fragpipe/
    ├── templates/                  # contains all workflow templates
    ├── per-plex/                   # contains all per-plex scripts
    ├── experiment-level-plex/      # contains all experiment-level scripts
    └── utility/                    # contains all utility scripts (FASTA gen, msconvert)
/home/maropakis.a/
└── ThermoRawFileParser/            # contains everything for TRFP

/scratch/maropakis.a/
├── Frag_outputs/                   # will be populated with all output information
├── Dependencies/
│   ├── sample_map/                 # contains all *_sample_map.xlsx
│   ├── FASTA_appended/             # contains all appended FASTAs from Decode output
│   ├── FASTA_fragpipe/             # contains all FragPipe-compatible appended FASTAs
│   └── mtp_maps/                   # will contain per-plex .csv files
├── AAS_Pipeline/                   # will contain all Decode output files
├── raw/                            # contains all raw files
├── spectra/                        # contains all .mzML spectra generated by msconvert.py
└── MQ_outputs/                     # contains all MaxQuant Dependent Peptide search outputs per plex

Workflow

1. Define your resolution unit -- single-plex (per-plex) or bridged (pipeline/experiment-level-plex)?
2. Choose the correct scripts in /Pipeline
3. (Optional) If starting from *_MTP.fasta appended with SAAP sequences from Decode output", run prepFASTA.py and build_FragFASTA.py (or, build_FragFASTA.sh)
4. (Optional) If already starting with a compatible FASTA, just mind the Path
5. Run the respective gen_fragpipe_*.py script, or the _run_.sh script as needed
6. Run FragPipe search!