HUMAnN2 is a system for accelerated functional profiling of shotgun metagenomic and metatranscriptomic (“meta’omic”) sequencing from host- and environmentally-associated microbial communities. HUMAnN2 implements a tiered search strategy comprised of three search phases (tiers). In the first search tier, the meta’omic sample is rapidly screened to identify known species in the underlying community. This information is then used to construct a custom gene sequence database for the sample by concatenating precomputied, functionally annotated pangenomes of detected species. In the second search tier, the entire sample is aligned against this database, yielding i) per-species, per-gene alignment statistics and ii) a collection of unmapped reads. In the final search tier, unmapped reads are aligned against a user-specified (typically comprehensive and nonredundant) protein database by translated search, yielding i) taxonomically unclassified per-gene alignment statistics and ii) a collection of novel reads. Per-gene alignment statistics are weighted based on alignment quality, coverage, and sequence length to yield gene abundance values i) for the community and ii) stratified according to per-species and “unclassified” contributions. Gene abundance values are finally applied to metabolic network reconstruction to identity and quantify pathways in the community (also stratified according to per-species and “unclassified” contributions). These processes, including the underlying databases and search parameters, are expanded in detail below.