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Dataset in Functional Dark Matter and Annotation Resolution, Lanthanide-Dependent Methylotrophy, Resistance, Defense Systems, and Mobile Elements, Subsurface and Soil Community Ecology

eggNOG

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Sources cited
+111 project reports and 2 cross-project digests, listed in the margin
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supports 7refines 9
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32 pages13 topics

What it is

Canonical name: eggNOG. 1

Known aliases: eggNOG; eggNOG-mapper is the associated annotation workflow named in the truly-dark-gene analysis. 1,2,3

Stable external identifier: No stable external identifier is reported in these documents. 1,2,3

eggNOG is an orthology and functional-annotation dataset used to characterize antimicrobial-resistance (AMR) clusters, compare orthologous-group (OG) enrichment, assess metabolic functions in lineage- and environment-associated genomes, identify plant-enriched gene families, classify prophage-associated gene modules, and provide partial clues for genes that remain hypothetical after reannotation. 1,2,4,5,6,3

Evidence across applications

Coverage, complementarity, and annotation gaps

  • The AMR pangenome analysis compared eggNOG annotations for 77K AMR clusters with an 86M-cluster pangenome baseline. 1
  • Bakta product annotations and eggNOG hits were present for 93.0% of AMR clusters, while 7.0% had Bakta annotations only; among sparsely annotated one-source clusters, 55.4% were singletons versus 34.6% of two-source clusters. 1
  • A 132.5M-gene-cluster comparison supports using eggNOG alongside Bakta rather than as a replacement: eggNOG had higher COG coverage (51% versus 8.2%), KEGG coverage (38.5% versus 17.3%), and Pfam coverage (63% versus 7.7%), whereas Bakta had higher GO coverage (15% versus 7.4%), product descriptions (71.2% versus 70.4%), and unique UniRef50 links covering 79.2%. 7
  • The eggNOG–Bakta union increased any-functional-annotation coverage to 77.3%, and Bakta rescued 11.2M clusters among 39.2M missed by eggNOG; only 33.3% of Bakta’s 17.6M distinct UniRef50 IDs existed in the KBase Data Lakehouse UniProt identifier table. This refines the earlier AMR result in which 93.0% of clusters had either annotation source, showing that apparent coverage depends on the annotation dimension and corpus. 7,1
  • In the truly-dark-gene analysis, eggNOG-mapper provided partial signal for 43.5% of the 6,427 genes classified as truly dark. The report identifies 2,551 eggNOG annotations for 2,551 unique truly dark gene clusters, corresponding to 43.5% of the 5,870 unique clusters. 3
  • eggNOG evidence was one component of a 12-dimensional clue matrix used to distinguish genes with no clues, sequence-identification clues, partial functional clues, or phenotype-only clues. The report places 711 truly dark genes, or 11.1%, in the tier with partial functional evidence from Pfam, COG, or eggNOG. Together, the partial-function tier and phenotype-only tier contain 2,314 genes considered the most promising for narrowing experimental hypotheses. 3
  • For one prioritized candidate, DvH/206658, eggNOG suggested “trehalose synthase” despite the gene retaining a hypothetical annotation. This extends the resource’s role from broad pangenome comparison to hypothesis generation for unresolved genes, while the partial coverage means such clues are not equivalent to validated functional assignments. 3
  • The same report contrasts eggNOG’s partial functional signal with sparse domain and pathway coverage: only 4.0% of truly dark genes had Pfam hits and 4.6% had KEGG KOs. 3

Functional categories and comparison-class dependence

  • Among AMR clusters with eggNOG annotations, COG V (defense mechanisms) was enriched 7.05-fold, comprising 14.9% of AMR clusters versus 2.1% of the baseline; COG P (inorganic ion transport) was enriched 1.93-fold, comprising 10.7% versus 5.6%; and COG J (translation) was enriched 1.50-fold. COG L (replication), COG I (lipid metabolism), and COG N (cell motility) were depleted to 0.12-fold, 0.10-fold, and 0.08-fold, respectively. 1
  • A 32-species, 357,623-gene comparison supports the broader interpretation that conserved and novel gene classes have distinct functional profiles: novel or singleton genes were enriched in COG L (mobile elements) by +10.88%, COG V (defense mechanisms) by +2.83%, and COG S (unknown function) by +1.64%, while core genes were depleted in COG J by -4.65%, COG F by -2.09%, COG H by -2.06%, COG E by -1.81%, and COG C by -1.75%. 8
  • This cross-species result refines the AMR-specific COG L result: COG L was depleted to 0.12-fold in AMR clusters but enriched among novel or singleton genes by +10.88%, indicating that functional distributions are dependent on the comparison class rather than universally directional. 1,8
  • The cross-species analysis found the COG L enrichment consistent across species and interpreted it as evidence that mobile elements contribute substantially to genomic novelty; however, approximately 70% of genes had COG annotations, so unassigned genes may skew the distributions. 8
  • Composite COG assignments were counted once per gene rather than split among component letters. The LV composite showed +0.34% enrichment with 76% consistency and was interpreted as a possible multifunctional mobile-defense module. This qualifies category-level interpretation because eggNOG v6 annotations may differ from original COG assignments. 8

OG enrichment, pathways, and marker calibration

  • In the Bacillota_B comparison, per-OG Fisher’s exact tests across 14,109 Firmicutes-level OGs compared 10 deep-clay anchor genomes with 62 soil-baseline genomes using Benjamini–Hochberg false-discovery-rate (FDR) correction, a fold-difference threshold of ≥3, and a minimum of ≥3 anchor genomes positive. This identified 547 OGs enriched at q<0.05, strongly supporting the preregistered prediction of at least 10 enriched OGs. 2
  • This application refines the AMR workflow’s broad eggNOG functional categories: keyword scans classified 42 enriched OGs as anaerobic-respiration functions, 24 as sporulation-revival functions, and 12 as mineral-attachment or exopolysaccharide functions, while manual inspection indicated that the 462-OG “other or unannotated” category contained additional electron-transfer and anaerobic-respiration signal. 2
  • eggNOG cluster-level annotation propagation within ≥90% average-nucleotide-identity (AAI) clusters can miss strain-level marker variants, including a single non-functional dsrA in an otherwise complete operon. This qualifies interpretation of OG- and keyword-level functional counts in the deep-clay analysis. 4
  • The deep-clay report proposes testing all standard amino-acid-biosynthesis EC numbers in eggNOG as a finer-grained sensitivity analysis of the 18-pathway GapMind completeness metric. This extends eggNOG’s use from OG categorization toward resolving pathway-completeness ceiling effects. 4
  • Annotation-proxy results qualify eggNOG-based marker interpretation: Preferred_name='lanM' produced 505 additional hits with zero overlap with 62 Bakta-validated Lanmodulin genomes, eggNOG KO K02030 produced 46,369 nonspecific hits, and only 418 of 5,092 genomes with any xoxF marker hit both eggNOG K00114 and Bakta lanthanide-dependent methanol-dehydrogenase products. Marker definitions should therefore be reported explicitly rather than treating generic KOs or stale preferred names as definitive. 7
  • The lanthanide methylotrophy atlas refines this calibration: among 134,578 hit-bearing rows, Preferred_name='lanM' had 505 eggNOG-only calls versus 62 Bakta-only Lanmodulin calls, while eggNOG K02030 had 46,369 eggNOG-only calls; the report therefore uses Bakta as the source of truth for Lanmodulin and xoxJ, eggNOG K00114 as primary for xoxF with Bakta as a union source, and eggNOG K14028 as primary for mxaF. 9
  • The same atlas supports using eggNOG for broad marker discovery but qualifies functional interpretation: K00114 identified 3,690 xoxF genomes and K14028 identified 195 mxaF genomes, whereas 2,320 xoxF-bearing genomes initially lacked eggNOG PQQ-biosynthesis annotations; 1,472 had partial eggNOG pqq calls, 33 had complete pqqA-E calls, and 897 had no PQQ evidence from either eggNOG or Bakta. 9

Environmental, defense, and pathway-scale applications

  • In 108 ENIGMA samples, eggNOG-derived COG-fraction proxies were used to estimate site defense and stress scores from pangenome-linked community data. This extends eggNOG’s environmental use but qualifies broad-category interpretation: confirmatory genus-level contamination–defense tests were null, while coverage-adjusted exploratory defense associations were sensitive to mapping mode, covariates, taxonomic resolution, and FDR. 10
  • The ENIGMA analysis therefore refines the earlier evidence that eggNOG categories can reveal defense-enriched content: broad COG proxies did not establish a robust contamination-linked functional shift, and the report recommends curated metal-stress gene sets and pathway-level summaries for follow-up. 10
  • In the pan-bacterial defense analysis, eggNOG description-based CRISPR-Cas detection produced 96% species prevalence, whereas the specific Cas1 Pfam marker PF01867 produced approximately 55% prevalence on the same pangenome. This refines the marker-calibration caveats: the 96% value is treated as an upper bound, and CRISPR-Cas comparisons must specify their detection method. 11
  • The combined EggNOG-or-Pfam defense-presence set prioritized recall for syndrome analysis; this qualifies interpretation of CRISPR-Cas syndrome specificity even though the report did not expect the significance results to be invalidated. 11
  • The PHB-granule analysis supports eggNOG’s use for pathway-scale pangenome censuses: 118,513 PHB-related gene clusters were identified across 19,496 species, including 86,318 phaA clusters, 11,792 phaC clusters, 9,617 phaB clusters, 6,130 phaP clusters, and 4,656 phaZ clusters; no phaR clusters were identified. 12
  • This result qualifies interpretation of partial pathway calls: the 46.5% “precursors-only” category likely overestimates PHB capability because phaA and phaB also participate in general fatty-acid and thiolase metabolism, and the apparent absence of phaR may reflect incomplete annotation or misassignment of K18080. 12
  • The PHB analysis also qualifies domain-based interpretation: all 11,792 phaC clusters were classified as “other_pfam” because eggNOG domain fields contained names such as Abhydrolase_1 and PhaC_N rather than the expected Pfam accession IDs PF00561 and PF07167; mapping those names to the accessions is required for PHA synthase class analysis. 12
  • In the plant microbiome analysis, eggNOG was used with pangenome architecture, InterProScan, GapMind, NMDC, MGnify, and host metadata to test plant-associated gene-family enrichment across 293,059 GTDB r214 genomes. Of 5,671 eggNOG ortholog groups passing the 5% prevalence filter, 5,341 (94.2%) were significant in raw plant-versus-non-plant tests and 3,840 had OR > 2; this qualifies raw enrichment as largely taxonomic rather than necessarily plant-specific. 5
  • After phylum-level control, 50 enriched gene families retained significance; COG3569 had Fisher OR = 8.92 and phylogenetically controlled OR = 6.01, with q = 7.7e-242, and occurred in 54% of plant-associated species versus 12% of non-plant species. A per-species logistic regression controlling for plant status, log₁₀ genome size, and top-10 phylum dummies converged for 48/50 families; all 48 retained positive plant-association coefficients with absolute coefficient > 0.2 and BH-FDR q < 0.05, with controlled odds ratios for the top ten ranging from 4.4 to 6.6 and q-values reaching 10⁻⁹⁶. 5
  • All 50 enriched families had eggNOG descriptions and InterProScan domains, 48/50 had GO terms, and 39/50 had MetaCyc pathways. Their dominant themes were unknown-function COG category S (19/50, or 38%), electron transport and energy metabolism (7/50, or 14%), inorganic ion transport (6/50, or 12%), carbohydrate metabolism (4/50, or 8%), and secondary metabolite biosynthesis (3/50, or 6%). This supports eggNOG’s use for controlled discovery of plant-associated functions while leaving causal direction unresolved. 5
  • The plant analysis refines the earlier coverage and category caveats: COG3569 and other retained families may contribute to rhizosphere or microaerobic competitiveness, but the raw 5,341/5,671 signal, two singular models (COG2351 and COG5343), and incomplete phylogenetic control prevent treating these enrichments as definitive plant adaptations. 5
  • In a prophage ecology analysis, eggNOG annotations identified prophage-associated modules across 27,702 bacterial species and 4,005,537 annotated gene clusters: packaging and lysogenic regulation occurred in 100.0% of species, lysis in 99.9%, integration in 99.1%, anti-defense in 64.3%, head morphogenesis in 56.1%, tail in 55.6%, and all 7 modules in 34.9%. This extends eggNOG-based pangenome annotation from broad functional categories to module-level viral ecology. 6
  • The prophage analysis supports environmental and structural interpretation of eggNOG module calls: packaging, lysis, and lysogenic-regulation genes were more consistently co-localized, whereas integration and anti-defense genes were more dispersed; environmental enrichment was concentrated in tail, head morphogenesis, and anti-defense modules. 6
  • This result qualifies the apparent near-universal prophage prevalence because the calls were based on eggNOG functional annotations rather than dedicated prophage detectors; domesticated remnants, bacterial homologs, and bacterial integrases may therefore contribute, and the false-positive rate was uncharacterized. 6
  • The same study supports using eggNOG annotations in cross-collection analyses: pangenome module enrichments agreed with NMDC metagenomic correlations for head morphogenesis, tail, and anti-defense, although NMDC prophage burden was inferred indirectly and had not been independently validated for prophage genes. 6

Integration and join constraints

eggNOG categories can distinguish conserved functions from mobile, defense, unknown-function, pathway-associated, and prophage-associated genome content, but the apparent signal depends on annotation coverage, marker definition, cluster propagation, detection method, comparison class, phylogenetic control, and environmental mapping resolution. These results support pangenome-integration while the truly-dark-gene analysis extends the integration target to residual hypothetical genes and the marker studies qualify any interpretation that treats an annotation clue as a validated function. 1,2,4,8,7,10,9,11,12,5,6,3

  • eggNOG annotation joins must respect cluster scope: eggnog_mapper_annotations.query_name joins to gene_cluster.gene_cluster_id, not gene.gene_id; cross-species gene-cluster identifiers are species-specific and should instead be compared through shared representations such as COG categories, KEGG orthologs, or Pfam domains. This refines cross-species COG interpretation by identifying the required integration keys. 13
  • The pitfalls report refines this integration guidance by requiring live schema inspection and cluster-level join validation before interpreting eggNOG annotations. 13

Tensions

  • eggNOG can provide useful partial clues for truly dark genes—43.5% of the 6,427 classified genes had partial signal, and DvH/206658 received a “trehalose synthase” clue—but marker and pathway applications show that generic KOs, propagated cluster calls, broad COG proxies, and annotation-based prophage calls can be nonspecific or incomplete. These are different evidence levels rather than a resolved contradiction: the former supports hypothesis generation, while the latter qualifies functional confirmation. 3,7,4,10,6

Open Directions

  • Re-run eggNOG joins after live schema inspection and verify cluster-level keys, then quantify how many apparent cross-species differences persist when represented through shared COG, KEGG, or Pfam identifiers. 13,8
  • Test all standard amino-acid-biosynthesis EC numbers against eggNOG and compare the result with the 18-pathway GapMind metric to identify pathway-completeness ceiling effects. 4
  • Validate high-priority truly-dark-gene clues, including the DvH/206658 trehalose-synthase hypothesis, with targeted sequence, biochemical, or genetic assays. 3
  • Reassess marker claims with explicitly defined unions and intersections of eggNOG, Bakta, Pfam, and dedicated detectors, especially for Lanmodulin, xoxF, mxaF, CRISPR-Cas, PHB genes, and prophage modules. 7,9,11,12,6
  • Replace broad COG environmental proxies with curated metal-stress and pathway-level gene sets, then repeat the ENIGMA contamination analysis with coverage adjustment, covariate control, taxonomic-resolution sensitivity, and FDR correction. 10
  • Map eggNOG domain names such as Abhydrolase_1 and PhaC_N to PF00561 and PF07167, and test whether the apparent absence of phaR reflects incomplete annotation or K18080 misassignment. 12
  • Repeat plant-associated OG enrichment with stronger phylogenetic control and experimental or comparative validation to determine whether COG3569 and other retained families represent plant adaptation rather than lineage structure. 5

Sources cited

  1. Pan-Bacterial AMR Gene Landscapeamr_pangenome_atlas · cited 15× · primary source · refines
  2. Subsurface Bacillota_B Specialization — What Distinguishes Deep-Clay Lineages from Soil Congeners?bacillota_b_subsurface_accessory · cited 8× · refines
  3. Truly Dark Genes — What Remains Unknown After Modern Annotation?truly_dark_genes · cited 14×
  4. Self-Sufficiency, Anaerobic Toolkit, and Cultivation Bias in Clay-Confined Cultured Bacterial Genomesclay_confined_subsurface · cited 7×
  5. Plant Microbiome Ecotypes — Compartment-Specific Functional Guilds and Their Genetic Architectureplant_microbiome_ecotypes · cited 9× · supports · refines
  6. Prophage Gene Modules and Terminase-Defined Lineages Across Bacterial Phylogeny and Environmental Gradientsprophage_ecology · cited 10× · supports
  7. Discoveries Logdiscoveries · cited 10× · cross-project digest · supports · refines
  8. COG Functional Category Analysiscog_analysis · cited 8× · supports · refines
  9. Lanthanide Methylotrophy Atlas — Summarylanthanide_methylotrophy_atlas · cited 6× · refines · supports
  10. Contamination Gradient vs Functional Potential in ENIGMA Communitiesenigma_contamination_functional_potential · cited 6× · refines
  11. Pan-Bacterial Anti-Phage Defense Arsenalphage_defense_arsenal · cited 6× · refines
  12. Polyhydroxybutyrate Granule Formation Pathways — Distribution Across Clades and Environmental Selectionphb_granule_ecology · cited 8× · supports
  13. KBase Data Lakehouse: Common Pitfalls & Gotchaspitfalls · cited 6× · cross-project digest · refines