Chapter 9: Functional Profiling & Metabolic Pathway Reconstruction with HUMAnN3
Chapter 9: Functional Profiling & Metabolic Pathway Reconstruction with HUMAnN3
Knowing who is in a microbial community is only half the story; understanding what they are doing requires functional profiling. HUMAnN3 (HMP Unified Metabolic Analysis Network 3) quantifies the abundance and coverage of metabolic pathways from metagenomic sequencing.
The HUMAnN3 Tiered Search Strategy
- Tier 1 (Fast Pangenome Search): Reads are aligned to species pangenomes identified by MetaPhlAn using Bowtie2.
- Tier 2 (Translated Search with Diamond): Unmapped reads are translated into protein frames and searched against UniRef50/UniRef90 to capture uncharacterized orthologs.
- Pathway Reconstruction: Maps detected enzymes to MetaCyc pathways, calculating Pathway Abundance and Pathway Coverage.
1. Running HUMAnN3
# Run HUMAnN3 functional quantification
humann \\
--input decontaminated_reads.fastq.gz \\
--output ./humann3_out/ \\
--threads 16 \\
--protein-database /ref/uniref90/ \\
--nucleotide-database /ref/chocophlan/
# Outputs generated:
# ├── sample_genefamilies.tsv -> Abundance of UniRef90 gene families (in RPK)
# ├── sample_pathabundance.tsv -> Abundance of MetaCyc pathways
# └── sample_pathcoverage.tsv -> Completeness (0.0 to 1.0) of each pathway