Metagenome Functional Profiling

Workflow

1. If HUMAnN: run humann with --input FASTQs, optionally with --taxonomic-profile for guided search.
2. HUMAnN produces: gene families (RPK), pathway abundance, pathway coverage.
3. Normalize HUMAnN output with humann_renorm_table (CPM or relative abundance).
4. If Prokka: run on contigs/MAGs for gene prediction and annotation.
5. If eggNOG-mapper: run emapper.py on predicted protein sequences for COG/KEGG/GO annotation.
6. Report: number of gene families, pathways detected, top abundant pathways, functional category distribution.

Output Contract

• HUMAnN gene family table — Gene families with RPK values (<outdir>/<sample>_genefamilies.tsv).
• HUMAnN pathway abundance table — Pathway abundances (<outdir>/<sample>_pathabundance.tsv).
• HUMAnN pathway coverage table — Pathway coverage scores (<outdir>/<sample>_pathcoverage.tsv).
• Prokka annotations — GFF, GBK, FAA, FFN files (<outdir>/prokka/).
• eggNOG annotation table — COG, KEGG, GO annotations (<outdir>/eggnog/<sample>.emapper.annotations).
• Functional summary — Top pathways, gene family counts, functional category distribution (<outdir>/functional_summary.txt).

Limits

• HUMAnN 3 requires ChocoPhlAn (approximately 15 GB) and UniRef90 (approximately 20 GB) databases.
• Prokka is designed for prokaryotic genomes; use appropriate --kingdom for archaea.
• eggNOG database is approximately 45 GB.
• HUMAnN is computationally intensive and may take hours per sample; for large cohorts, consider running on HPC.
• HUMAnN, Prokka, and eggNOG-mapper must be installed and available on $PATH.
• Prokka and eggNOG-mapper operate on contigs/proteins, not raw reads.
• Common failure cases:
  • HUMAnN ChocoPhlAn or UniRef databases not downloaded or path misconfigured.
  • Prokka failing on non-prokaryotic contigs when --kingdom is set incorrectly.
  • eggNOG-mapper database version mismatch with the installed emapper version.