Workflow Details

What FBMN Actually Does

Your data goes through these stages in order:

1. Metadata merge — Your sample metadata is loaded and linked to file names.
2. Input validation — Column names and file formats are checked; warnings are logged but the pipeline continues.
3. Quantification reformatting — Your feature table (MZmine/XCMS/MS-DIAL format) is converted to the internal GNPS format.
4. Spectra filtering — Peaks within the precursor isolation window are removed, and a noise filter is applied. This improves cosine score quality.
5. Molecular networking — Every pair of spectra is compared by cosine similarity. Pairs above the threshold become edges.
6. Edge filtering — Each node keeps only its top-K best edges; components exceeding the maximum size are pruned.
7. Library search — Each spectrum is compared against the local GNPS spectral library.
8. Cluster info summary — Features, quantification values, and metadata are merged into a single table.
9. Enrichment — Library hits and component indices are added to the cluster info table.
10. GraphML export — Everything is assembled into a single file readable by Cytoscape.

Key Parameters and When to Change Them

Parameter	Default	What it does	When to change
Cosine Score Threshold	0.7	Minimum similarity for an edge	Lower to 0.5 for exploratory analysis; raise to 0.85 for validation
Min Matched Peaks	6	Minimum shared fragment ions for an edge	Lower to 4 for low-quality spectra; raise to 8 for high confidence
Top K Edges per Node	10	Max neighbors per feature	Lower for cleaner networks; raise for community detection
Max Component Size	100	Largest allowed cluster	Set to 0 (unlimited) if you want to see full propagation
Fragment Tolerance	0.02 Da	m/z window for matching fragment ions	Raise to 0.05 for ion-trap data; keep at 0.02 for Orbitrap/Q-TOF

Current Limitations

• Analog search is implemented in the pipeline but awaiting verification.
• Statistical analysis (volcano plots, PCoA) requires the RUN_STATS parameter and is not yet enabled in the UI.
• QIIME2 integration and ili 3D spatial mapping are not available in the local version.
• Only one MGF file is supported per job (multi-file input must be merged upstream).