bigbio/relink

Introduction

bigbio/relink is a bioinformatics pipeline for crosslinking mass spectrometry (XL-MS) data analysis. It processes mass spectrometry data through several stages including file conversion, linear search for mass recalibration, mass recalibration, crosslinking search, and FDR correction using the xiSEARCH/xiFDR suite.

The pipeline is built using Nextflow, a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It uses Docker/Singularity containers making installation trivial and results highly reproducible. The pipeline follows nf-core guidelines.

Pipeline Summary

1. File Conversion - Convert Thermo RAW files to MGF format using ThermoRawFileParser
2. Linear Search - Run xiSEARCH linear peptide search for mass error estimation
3. Mass Recalibration - Calculate and apply mass corrections to MS1 and MS2 spectra
4. Crosslinking Search - Run xiSEARCH crosslinking peptide search
5. FDR Correction - Apply xiFDR for false discovery rate estimation
6. Reporting - Generate MultiQC report with analysis summary

Quick Start

1. Install Nextflow (>=23.04.0)

2. Install any of Docker, Singularity (you can follow this tutorial), Podman, Shifter or Charliecloud for full pipeline reproducibility

3. Download the pipeline and test it on a minimal dataset with a single command:

   nextflow run bigbio/relink -profile test,docker --outdir ./results

4. Start running your own analysis:

   nextflow run bigbio/relink \
       -profile docker \
       --input samplesheet.csv \
       --outdir ./results

Input Samplesheet

The input samplesheet is a CSV file with the following columns:

sample,file,fasta,xi_linear_config,xi_crosslink_config
sample1,/path/to/sample1.raw,/path/to/database.fasta,/path/to/xi_linear.conf,/path/to/xi_crosslinking.conf
sample2,/path/to/sample2.raw,/path/to/database.fasta,/path/to/xi_linear.conf,/path/to/xi_crosslinking.conf

Column	Description
sample	Sample identifier (unique)
file	Path to RAW or MGF file
fasta	Path to FASTA database
xi_linear_config	Path to xiSEARCH linear configuration file
xi_crosslink_config	Path to xiSEARCH crosslinking configuration file

Parameters

Input/Output Options

Parameter	Description	Default
--input	Path to input samplesheet CSV	Required
--outdir	Output directory for results	./results

Analysis Options

Parameter	Description	Default
--do_recalibration	Perform mass recalibration	true
--do_crosslinking_search	Perform crosslinking search	true
--do_fdr	Perform FDR correction	true
--do_mass_error_plots	Generate mass error plots	false
--link_fdr	Link-level FDR threshold (%)	5

Output

The pipeline outputs the following directories:

results/
├── mgf/                    # Converted MGF files
├── linear_search/          # Linear search results
├── recalibrated/           # Recalibrated MGF files
│   └── plots/              # Mass error plots (optional)
├── crosslinking_search/    # Crosslinking search results
├── fdr/                    # FDR-corrected results
├── multiqc/                # MultiQC report
└── pipeline_info/          # Pipeline execution info

Software Used

• ThermoRawFileParser - RAW file conversion
• xiSEARCH - Crosslinking peptide search
• xiFDR - FDR estimation
• pyOpenMS - Mass spectrometry data processing
• Polars - Data processing

Citations

If you use bigbio/relink for your analysis, please cite:

xiSEARCH: Mendes, M.L., et al. (2019). "An integrated workflow for crosslinking mass spectrometry." Molecular Systems Biology.

xiFDR: Fischer, L., & Rappsilber, J. (2017). "Quirks of error estimation in cross-linking/mass spectrometry." Analytical Chemistry.

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

Contributing

We welcome contributions! Please see our contribution guidelines for details.

Support

For questions, issues, or feature requests, please open an issue on the GitHub repository.

License

This pipeline is released under the Apache 2.0 License.