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Workflow: pxd_to_speclib

Snakemake workflow that reanalyzes proteomics data from a PRIDE Archive project to create a spectral library.

The workflow goes through the following steps:

  • Download RAW files from PRIDE Archive for given PXD identifier
  • Convert RAW files to MGF using the CompOmics ThermoRawFileParser
  • Search with MSGFPlus
  • Generate Percolator input files
  • Postprocess search results with Percolator
  • Parse results to generate a spectral library

Requirements

  • Conda (tested on Linux)

Run the workflow

  1. Create and activate the environment:
conda env create -f envs/pxd_to_speclib.yml
conda activate pxd_to_speclib

  1. Setup your configuration:

    • conf/snakemake_config.json (see Configuration)
    • conf/msgfplus_params.txt
    • Add required input files (e.g. fasta sequence database)

  2. Run the workflow:

    • To create a general spectral library: snakemake . --use-conda
    • To create a calibrated retention time dataset: snakemake --snakefile make_rt_lib.smk --use-conda

Configuration

All settings can be found in a JSON configuration file: conf/snakemake_config.json.

Section Option Default value Description
download pxd_identifier "PXD000000" PXD identifier of PRIDE Archive project to download.
file_pattern ".*" Regular expression that matches all raw file filenames to download (.* matches all filenames).
convert exec "ThermoRawFileParser.sh" Executable command to call ThermoRawFileParser. See Note 1.
search msgfplus_conf "conf/msgfplus_params.txt" Path to MSGFPlus configuration file.
fasta "path/to/search_db.fasta" Path to protein fasta. Important: MSGFPlus will add decoy peptides by default; they should not yet be present in the given fasta file.
msgfplus_exec "msgf_plus" Executable command to call MSGFPlus. See Note 2.
threads_per_search 5 Number of threads per MSGFPlus search. See Note 3.

Note 1

ThermoRawFileParser executable
To use a custom version of the ThermoRawFileParser, change convert > exec to:

  • Using environment TRFP: "ThermoRawFileParser.sh"
  • Using custom TRFP: "mono /path/to/ThermoRawFileParser.exe"

Note 2

MSGFPlus executable
Idem for MSGFPlus, change search > msgfplus_exec to:

  • Using environment MSGFPlus: msgf_plus
  • Using custom jar file: "msgfplus_exec": "java -Xmx5000M -jar /path/to/MSGFPlus/MSGFPlus.jar"

The latter allows a custom memory limit for the Java VM. By default, this is 1GB.

Note 3

Indirectly limit memory usage while searching with threads_per_search
The option search > threads_per_search defines the number of threads each individual search can use. In combination with the snakemake --cores x option, this allows you to constrict the number of parallel searches. E.g.: the combination of --cores 24 and threads_per_search: 6 limits the number of parallel searches to 4. This can be convenient if you would run into memory issues caused by too many parallel searches.