digestome: a licence-clean marker-gene panel for functional profiling of anaerobic digestion microbiomes

Functional profiling of anaerobic digestion microbiomes is routinely performed against KEGG or MetaCyc, whose paid commercial licences block fee-for-service analysis for biogas operators, the setting where the results have the most immediate operational value. We present digestome, a curated marker-gene panel for anaerobic digestion built exclusively on sources that are free for commercial use (NCBIfam, public domain; Pfam, CC0; Rhea, CC BY), together with a scorer that reports pathway completeness, branch capability and, for hydrolysis, whether the enzyme is built for export. Benchmarked against 1,401 metagenome-assembled genomes from 134 anaerobic digesters, the panel assigned no methanogenesis route to any of 1,361 non-methanogens, and every acetoclastic call fell within Methanosarcina or Methanothrix. The result held for 3,043 species representatives from the Genome Taxonomy Database, spanning 203 phyla, which are built differently from binned metagenomes: the only background genomes given a route were two archaea carrying mcrA, and none of the 90 anaerobic methane and alkane oxidisers was given one. Specificity against non-methanogens does not show that a methanogen gets the right route: with a family-level check, 59 of 156 acetoclastic calls in that sample fell on methylotrophic genera that do not use acetate, and a lineage policy removed them, with 14 more left unconfirmed in unnamed genera. Testing whether a catalytic domain shares a polypeptide with an export module reduced the genomes credited with cellulolytic capacity from 597 to 50. A causal graph of the measurement shows that a profile reports the community together with three measurement choices (sampling, DNA extraction and sequencing depth, and reference genomes), which fixes what one profile can attribute and what a prospective validation would have to record.