Bio Comparative Genomics Genome Distance And Species Delineation

Version Compatibility

Reference examples tested with: skani 0.2.5+ (Shaw & Yu 2023 Nat Methods 20:1661; bluenote-1577/skani), FastANI 1.34+ (Jain 2018 Nat Commun 9:5114), pyani 0.3.0+ (Pritchard 2016 Anal Methods 8:12), pyskani 0.1+ (Larralde 2025), OrthoANI 1.40+ (Lee 2016 Int J Syst Evol Microbiol 66:1100), OrthoANIu 1.2+, GTDB-Tk 2.7.1+ (Chaumeil 2022 Bioinformatics 38:5315), GTDB release 220 (2024-Q3+), TYGS web (Meier-Kolthoff & Goker 2019 Nat Commun 10:2182), GGDC v3.0 (web), Mash 2.3+ (Ondov 2016 Genome Biol 17:132), Dashing 2 (Baker & Langmead 2023 Genome Res 33:1218), CompareM 0.1.2+ for AAI (Parks/Cherubini), pyANI 0.3.1+, BLAT 36+, DIAMOND 2.1+. JSpeciesWS web (Richter et al 2016 Bioinformatics 32:929).

Before using code patterns, verify installed versions match. If versions differ:

• CLI: skani --version; fastANI --version; gtdbtk --version; mash --version; pyani --version
• Python: pip show gtdbtk pyani

If code throws GTDB-Tk database not found, skani sketch incompatible, Mash sketch version, these tools have database-version coupling: GTDB-Tk requires the GTDB release matched to the binary version; Mash/skani sketches are forward-compatible but not always backward. Check gtdbtk check_install for database completeness.

Genome Distance and Species Delineation

"Are these genomes the same species, and what species are they?" -> Prokaryote species delineation has shifted from 16S rRNA identity (now considered insufficient at < 98.7%) to whole-genome ANI at a 95% threshold (Jain 2018 Nat Commun 9:5114; corroborating Goris 2007 and Konstantinidis 2005). The modern operational standard for taxonomy is GTDB-Tk (Chaumeil 2020/2022 Bioinformatics 38:5315), which assigns genomes to the Genome Taxonomy Database (GTDB) using ANI radius + marker-gene placement. skani (Shaw & Yu 2023 Nat Methods 20:1661) has replaced FastANI as the default ANI tool in GTDB-Tk 2.4+ for being 20-30x faster while maintaining accuracy. The 95% ANI threshold is robust but not absolute -- the species circumscription radius varies by genus (Parks 2018 Nat Biotech 36:996).

• CLI: skani dist genomes1.fa genomes2.fa -t 16 -- fast ANI computation
• CLI: fastANI -q query.fa -r reference.fa -o output.txt -- standard ANI
• CLI: gtdbtk classify_wf --genome_dir genomes/ --out_dir gtdbtk_out --cpus 32 -- GTDB classification
• CLI: mash dist *.fa -- k-mer MinHash distance
• Web: TYGS (https://tygs.dsmz.de/) and GGDC (https://ggdc.dsmz.de/) for dDDH

Algorithmic Taxonomy

Methodology evolves; verify GTDB release (currently r220 / 2024-Q3) and GTDB-Tk version compatibility. The 95% ANI species threshold has been confirmed across 90,000+ prokaryote genomes (Jain 2018; Parks 2018 demonstrating clear bimodality).

Decision Tree by Experimental Scenario

Per-Tool Failure Modes

skani / FastANI ANI saturating below 75%

Trigger: Computing ANI between two genomes at < 75% nucleotide identity.

Mechanism: ANI is computed only on alignable regions; at < 75% identity, alignment fraction drops dramatically (< 50%); the few alignable regions are biased toward conserved regions, inflating apparent ANI.

Symptom: Reported ANI 75-80% with alignment fraction < 50%; meaningless biologically.

Fix: Below 75% ANI, switch to AAI (amino-acid identity from translated proteins); AAI is more meaningful at deep divergence (~50% AAI between distant genera). For ANI matrix at long range, use Mash distance (k-mer-based, no alignment).

GTDB-Tk database version mismatch

Trigger: Using GTDB-Tk binary with mismatched GTDB reference data version.

Mechanism: GTDB releases (r207, r214, r220, ...) include reference trees, marker-gene HMMs, and ANI sketch files; GTDB-Tk versions are aligned to specific releases. Mismatch causes silent or loud failures.

Symptom: GTDB-Tk fails with "marker gene HMM not found" or runs but produces inconsistent classifications.

Fix: Check release compatibility: gtdbtk check_install shows the expected vs found versions. Pin via conda env. Download the matching release from data.gtdb.ecogenomic.org.

Below-95% ANI but same species (genus-specific radius)

Trigger: Strict 95% ANI threshold; rejecting closely related genomes as different species.

Mechanism: Parks 2018 demonstrated species-circumscription radius varies (typically 95-99% but tighter for some clonal lineages). For some genera (e.g., Pseudomonas), the species threshold is 94% per genus-specific analysis.

Symptom: Two clearly biologically-related strains (epidemiologically connected outbreak) have ANI 94%; using strict 95% calls them different species.

Fix: Use GTDB-Tk's species radius approach which uses genus-specific cutoffs. For novel-species naming, consider 95% as primary + ecology + biology. Report ANI alongside additional context (gene content, phenotype, ecology).

High alignment fraction required (AF >= 0.5)

Trigger: ANI reported without alignment fraction; or AF < 0.5.

Mechanism: A 95% ANI with 20% AF (only 20% of genome alignable) is biologically meaningless; the comparison covers a small fraction of the genomes. Standard species delineation requires AF >= 0.5 + ANI >= 95%.

Symptom: ANI calls "same species" but only 20-30% of genome aligned.

Fix: Require AF >= 0.5 + ANI >= 95% for species call. Below this, classification is ambiguous; consider AAI or HGT (e.g. a phage-rich genome will have low AF to its actual species).

MAG contamination / incompleteness

Trigger: Running GTDB-Tk on MAGs without CheckM2 pre-screening.

Mechanism: Low-completeness MAGs (< 50%) may have marker-gene gaps that break GTDB-Tk's placement; high-contamination MAGs (> 5%) place anomalously.

Symptom: GTDB-Tk reports "no genome retained" or makes implausible classifications.

Fix: Pre-filter MAGs with CheckM2 (Chklovski 2023 Nat Methods 20:1203): require >= 70% completeness, < 5% contamination, < 10% strain heterogeneity for species-level. For lower-quality MAGs, report genus-level only or exclude.

Mash distance vs ANI inconsistency

Trigger: Using Mash distance for species delineation directly.

Mechanism: Mash distance = 1 - (Mash similarity) is correlated with 1 - ANI but is not the same metric. The 0.05 Mash distance threshold (sometimes cited as "5% Mash = 95% ANI") is approximate; exact ANI varies +-0.5%.

Symptom: Genomes with Mash distance 0.04-0.06 inconsistently called "same species" or not.

Fix: Use Mash for fast screening + clustering, but verify species delineation with ANI on candidate pairs. Mash 0.05 ~ ANI 95% is a rough heuristic; not a publishable threshold.

Ortholog-based ANI conservative vs alignment-based

Trigger: Comparing OrthoANI / OrthoANIu to FastANI / skani.

Mechanism: OrthoANI uses reciprocal-best-orthologs; FastANI uses fragment alignment. They differ by 0.5-2% systematically; OrthoANI is more conservative.

Symptom: OrthoANI = 94.5%, FastANI = 96% on same pair; different "same species" calls.

Fix: Document tool used; for taxonomy, use the tool aligned to the reference database (GTDB-Tk uses skani; NCBI uses FastANI). Cross-validation for unclear cases.

Tetranucleotide bias inflating Mash for low-GC genomes

Trigger: Comparing genomes with extreme GC content (Streptomyces ~70% vs Mycoplasma ~25%) via Mash.

Mechanism: k-mer frequency distributions are GC-dependent; Mash distance between extreme-GC genomes is inflated by background composition rather than biology.

Symptom: Two unrelated extreme-GC genomes (e.g. Mycoplasma + Mycoplasma) appear closer than they biologically are.

Fix: Use ANI / AAI for cross-GC comparisons; Mash is reliable only within a GC-comparable range. Document GC range when reporting Mash distances.

Type strain conflicts in TYGS

Trigger: Submitting genome to TYGS where type strain is missing or misclassified.

Mechanism: TYGS depends on type strain database; if the type strain is genome-sequenced incompletely or misclassified, the placement may be incorrect.

Symptom: TYGS reports unexpected nearest type strain.

Fix: Cross-validate with GTDB-Tk; check type-strain genome quality. For novel-species naming, TYGS report should be supplemented with manual taxonomic check.