Bio Comparative Genomics Ortholog Inference

Version Compatibility

Reference examples tested with: OrthoFinder 3.0+ (bioRxiv 2025.07.15.664860), SonicParanoid 2.0.8+ (Cosentino 2023), Broccoli 1.2+ (Derelle 2020), ProteinOrtho 6.3.0+ (Lechner 2011 + recent), OMA standalone 2.6.0+, FastOMA 0.3.5+ (Majidian 2025), eggNOG-mapper 2.1.12+, JustOrthologs 2.0+, DIAMOND 2.1.10+, MMseqs2 17-b804f+, IQ-TREE 2.3.6+, BUSCO 5.7+, Compleasm 0.2.7+, BioPython 1.84+, R 4.4+ for downstream tree-based reconciliation.

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

• CLI: orthofinder --help; sonicparanoid --help; oma --help
• Python: pip show eggnog-mapper; which fastoma

If code throws Diamond requires N more sequences than provided, KeyError on species tree taxa, STAG branch length 0, or HOG file format mismatch, the OrthoFinder v2 -> v3 file layout changed (Orthogroups/ -> Phylogenetic_Hierarchical_Orthogroups/; rooted gene trees are now per-HOG); update parsing accordingly.

Ortholog Inference

"Find the orthologs of my gene(s) across these species" -> Choose between graph-based (RBH / similarity-clustering: fast, lower recall) and tree-based (gene-tree reconciliation: higher accuracy, slower) frameworks; recognize that "orthology" splits into 1:1, 1:many, many:many, and the practical unit for most pipelines is the HOG (Hierarchical Orthologous Group) -- a maximal cluster of genes descended from a single ancestral gene at a defined taxonomic level (Altenhoff 2013 PLoS Comp Biol 9:e1002954). The "ortholog conjecture" (orthologs more functionally similar than paralogs) is supported but weakly (Altenhoff 2012 PLoS Comp Biol 8:e1002514); don't treat 1:1 ortholog labeling as automatic functional equivalence.

• CLI: orthofinder -f proteomes/ -t 16 -M msa -- HOG output in v3 layout
• CLI: sonicparanoid -i proteomes/ -o output --mode default -- ML predictor + protein language model
• CLI: broccoli.py -dir proteomes/ -threads 16 -- direct OG with chimeric handling
• CLI: oma standalone HOG inference at every taxonomic level
• CLI: proteinortho6.pl --project=run proteomes/*.faa -- graph clustering with optional synteny
• CLI: emapper.py -i proteins.faa --output project --cpu 16 -- eggNOG annotation transfer

Algorithmic Taxonomy

Methodology evolves; Quest-for-Orthologs benchmarks (Altenhoff 2024 Genome Biol 25:115) refresh annually. Verify the current QfO benchmark results before locking on a single tool for novel benchmarking-grade work.

Decision Tree by Experimental Scenario

Per-Method Failure Modes

Hidden paralogy from missing outgroup

Trigger: OrthoFinder run without a sufficiently distant outgroup; only ingroup taxa included.

Mechanism: OrthoFinder roots gene trees via STRIDE / STAG using outgroup-based duplication signals. Without an outgroup, the root inferred for each gene tree may be internal, mistakenly classifying a duplication-then-loss pattern as orthology. The "1:1 orthologs" returned may actually be hidden paralogs (Emms & Kelly 2017 MBE 34:3267 STRIDE; benchmark shows 8% improvement in HOG accuracy with outgroup).

Symptom: Phylogenomic concatenation produces low-bootstrap species tree; per-gene trees show inconsistent rooting; same-clade species show longer-than-expected branches in single-copy ortholog trees.

Fix: Include >= 1 outgroup taxon at the next-higher taxonomic level (sister phylum / class / order). For vertebrates, use cyclostomes (lamprey) as outgroup for jawed vertebrates; for plants, use a non-flowering plant for angiosperms. Re-run with outgroup; cross-check HOG file Phylogenetic_Hierarchical_Orthogroups/N0.tsv for 1:1 stability.

Splice isoforms inflating copy number

Trigger: Proteome FASTA contains multiple isoforms per gene (Ensembl, RefSeq with -NR flag).

Mechanism: OrthoFinder / SonicParanoid / OMA treat each protein sequence as a gene; isoforms are clustered together within orthogroups but inflate the apparent gene count per species, producing artifactual "co-orthologs" that are just isoforms of one gene.

Symptom: Per-species gene count > 2x what gene-annotation pipeline reported; orthogroups contain multiple proteins from same gene; gene names contain isoform suffixes (.1, -iso1, etc.).

Fix: Pre-filter proteomes to longest isoform per gene. Use agat_sp_keep_longest_isoform.pl (AGAT toolkit), Biopython snippet on Ensembl gene-isoform mapping, or Trinotate longest-ORF picker. OrthoFinder3 ships a wrapper tools/primary_transcript.py for Ensembl/UniProt format.

Annotation heterogeneity inflating lineage-specific OGs

Trigger: Genomes annotated by different pipelines (Augustus, MAKER, Funannotate, NCBI RefSeq) mixed in one analysis.

Mechanism: Pipelines differ in handling of intron predictions, gene boundaries, and small-gene filtering. A gene predicted by Augustus but missed by MAKER appears as an apparent lineage-specific gene in the MAKER-annotated species, producing spurious "species-specific" orthogroups.

Symptom: CAFE (gene family evolution) shows extreme expansions / contractions for species with different annotation pipelines; per-species "unique" gene count varies 5-10x between technically similar genomes.

Fix: Re-annotate all genomes with one pipeline (e.g. BRAKER3 or Funannotate) before orthology. When re-annotation isn't possible, normalize via BUSCO/Compleasm completeness (filter OGs absent from species with < 95% BUSCO complete). Document annotation pipeline per species in methods.

Ortholog conjecture violations

Trigger: Transferring GO/KEGG functional annotation from 1:1 ortholog without testing functional divergence.

Mechanism: Studies show orthologs are weakly more functionally similar than paralogs (Altenhoff 2012 PLoS Comp Biol 8:e1002514; Stamboulian 2020 Bioinformatics 36:i219), but effect size is small and dependent on evolutionary distance. Subfunctionalization (Force 1999 Genetics 151:1531), neofunctionalization, and dosage subfunctionalization can rapidly differentiate 1:1 orthologs.

Symptom: Transferred annotation contradicts species-specific experimental data; ortholog has fold-change different expression patterns; rapid evolution (dN/dS > 0.3) on one branch only.

Fix: For high-confidence annotation transfer, require: (1) 1:1 orthology AND (2) low branch dN/dS (< 0.2 for both branches) AND (3) GO evidence code ISO with experimental support upstream. Tag annotation as "predicted from ortholog" rather than equating function. eggNOG ortholog-conjecture-aware mode provides confidence scoring.

RBH symmetric-but-wrong errors

Trigger: Reciprocal best hits (RBH) method on a gene that has been replaced by a paralog in one lineage.

Mechanism: Lineage A retains the original ortholog; Lineage B lost the ortholog and replaced its function with a paralog (xenologous replacement). RBH between A and B identifies the paralog as the "ortholog" because it's the best hit.

Symptom: dN/dS on RBH ortholog pair > 1 on the B branch (suggesting positive selection but actually paralog substitution); gene tree shows the B sequence is sister to other paralogs of the A gene, not to A.

Fix: Use tree-based methods (OrthoFinder3, OMA HOGs) for organisms with extensive paralog history. RBH is appropriate for orthogroup pre-screening only. JustOrthologs is explicitly an RBH method, so it's affected by this; reserve for closely related species.

Synteny ignored in WGD lineages

Trigger: Plant, yeast, fish, or salmonid analysis where one or more ancient WGD events occurred.

Mechanism: WGD doubles all genes; subsequent gene loss is biased toward certain functional categories (Dosage Balance Hypothesis; Freeling 2007 PNAS 104:8723). Sequence-only orthology cannot distinguish "ortholog" (single ancestral gene) from "homeolog" (paralog from WGD). In hexaploids (wheat, Brassica), this triples the confusion.

Symptom: Multiple co-orthologs per species in orthogroups for WGD-affected lineages; rate variation across "co-orthologs" suggests one is the true ortholog and the others are recent duplicates.

Fix: Use synteny-aware methods (GENESPACE -- Lovell 2022 eLife 78526; ProteinOrtho -synteny; OrthoFinder3 + post-hoc synteny verification). Restrict 1:1 ortholog phylogenomics to genes in single-copy syntenic regions. For deep WGD ancestry (e.g. 2R vertebrate WGD), modern orthologs of post-2R paralogs (ohnologs) can no longer be unambiguously identified by sequence; use synteny + duplication-dating.

MAFFT-only alignment in OrthoFinder MSA mode

Trigger: orthofinder -M msa -A mafft default; very divergent orthogroups (deep taxonomy).

Mechanism: MAFFT default settings choose accurate-but-fast iterative refinement. For sequences with >50% divergence, MAFFT's auto choice may downgrade to FFT-NS-2, producing alignments with > 30% poorly aligned columns that distort gene-tree inference and downstream HOG construction.

Symptom: Per-OG MAFFT logs show "FFT-NS-2 selected"; gene trees have unstable rooting; bootstrap support < 60% for many internal branches.

Fix: Force mafft-linsi or specify -A mafft --thread -1 --localpair --maxiterate 1000. For deeply divergent OGs, run PRANK or MUSCLE5 post-hoc on critical OGs and re-build gene trees with IQ-TREE. PREQUAL or HmmCleaner segment-filtering improves resulting trees (Di Franco 2019 BMC Eco Evo 19:21).

Quantitative Thresholds

OrthoFinder3 Standard Workflow

Goal: Produce HOG-based orthology with species tree and gene-duplication events for any clade.

Approach: Prepare cleaned per-species proteomes (longest isoforms) -> include outgroup -> run with MSA + IQ-TREE option -> parse HOG output at appropriate taxonomic level.

# Pre-clean proteomes: longest isoform per gene
for f in raw_proteomes/*.faa; do
    python tools/primary_transcript.py $f > cleaned/$(basename $f)
done

# OrthoFinder v3 with MSA + IQ-TREE for tree-based HOG inference
orthofinder \
    -f cleaned/ \
    -t 16 \
    -a 4 \
    -M msa \
    -A mafft \
    -T iqtree \
    -S diamond_ultra_sens \
    -y \
    -o orthofinder_run

# Output of interest (v3 layout):
# orthofinder_run/Results_<date>/Phylogenetic_Hierarchical_Orthogroups/N0.tsv  (root-level HOGs)
# orthofinder_run/Results_<date>/Single_Copy_Orthologue_Sequences/             (single-copy MSA-ready)
# orthofinder_run/Results_<date>/Species_Tree/SpeciesTree_rooted.txt
# orthofinder_run/Results_<date>/Gene_Duplication_Events/                       (per-branch dup counts)

'''Parse OrthoFinder v3 HOG output for downstream analysis.'''

import pandas as pd
from pathlib import Path


def load_hogs(results_dir, level='N0'):
    '''HOG file path differs from v2: Phylogenetic_Hierarchical_Orthogroups/{level}.tsv.'''
    p = Path(results_dir) / 'Phylogenetic_Hierarchical_Orthogroups' / f'{level}.tsv'
    df = pd.read_csv(p, sep='\t')
    return df.set_index('HOG')


def _is_present(v):
    '''OrthoFinder v3 HOG cells are either NaN (older versions), '' (newer), or a comma-separated gene list.'''
    return not (pd.isna(v) or v == '' or str(v).strip() == '')

def single_copy_hogs(hog_df, min_species_fraction=0.9):
    '''Return HOG IDs where every present species has exactly 1 gene and >= fraction of species present.'''
    sp_cols = [c for c in hog_df.columns if c not in ('OG', 'Gene Tree Parent Clade')]
    is_single = hog_df[sp_cols].apply(
        lambda r: all((not _is_present(v)) or ',' not in str(v) for v in r), axis=1
    )
    n_present = hog_df[sp_cols].apply(lambda r: sum(_is_present(v) for v in r), axis=1)
    keep = is_single & (n_present >= min_species_fraction * len(sp_cols))
    return hog_df.index[keep].tolist()


def classify_orthology(hog_df, sp_pair):
    '''Per-HOG classify pairwise orthology between two species.

    Returns one of: '1-1', '1-many', 'many-1', 'many-many', 'absent', 'sp1-only', 'sp2-only'.
    '''
    sp1, sp2 = sp_pair
    types = {}
    for hog, row in hog_df.iterrows():
        v1, v2 = row.get(sp1), row.get(sp2)
        n1 = 0 if not _is_present(v1) else len(str(v1).split(','))
        n2 = 0 if not _is_present(v2) else len(str(v2).split(','))
        if n1 == 0 and n2 == 0: types[hog] = 'absent'
        elif n1 == 0:           types[hog] = 'sp2-only'
        elif n2 == 0:           types[hog] = 'sp1-only'
        elif n1 == 1 and n2 == 1: types[hog] = '1-1'
        elif n1 == 1 and n2 > 1: types[hog] = '1-many'
        elif n1 > 1 and n2 == 1: types[hog] = 'many-1'
        else:                     types[hog] = 'many-many'
    return types

SonicParanoid2 + Cross-Validation

Goal: Run a second orthology method to cross-validate OrthoFinder3 calls (consensus increases QfO benchmark precision).

Approach: SonicParanoid2 with default ML predictor -> intersect orthogroups with OrthoFinder HOGs at the root level -> compute Jaccard agreement.

sonicparanoid -i cleaned/ -o sp2_run --mode default --threads 16 --pfam pre-computed
# Output: sp2_run/runs/<date>/orthogroups/flat.ortholog_groups.tsv

from itertools import combinations
import pandas as pd

def jaccard_ogs(ogs_a, ogs_b):
    '''Compute mean Jaccard between two orthogroup sets keyed by gene IDs.
    Each og is a frozenset of gene IDs.'''
    set_a = {gene: og for og in ogs_a for gene in og}
    set_b = {gene: og for og in ogs_b for gene in og}
    common = set(set_a) & set(set_b)
    jacs = []
    for g in common:
        a = set_a[g]
        b = set_b[g]
        j = len(a & b) / len(a | b) if a | b else 0
        jacs.append(j)
    return sum(jacs) / len(jacs) if jacs else 0

Consensus single-copy HOGs (1:1 in both methods) are the highest-confidence input for downstream phylogenomic concatenation or selection analyses.

TOGA for Vertebrate-Scale Orthology with Gene-Loss

Goal: Identify orthologs and classify gene-loss/intactness across hundreds of mammal or bird genomes.

Approach: Run Progressive Cactus whole-genome alignment (see [[whole-genome-alignment]]) -> TOGA projects reference genes through chains -> classifies each query gene as I/PI/UL/L/M/PM.

# After Cactus alignment producing reference-query chain files
toga.py \
    --chain cactus_chain.bb \
    --bed reference_genes.bed \
    --tDB target.2bit \
    --qDB query.2bit \
    --nextflow_dir nf_pipeline_dir \
    --pn project_name \
    --cpu 64 \
    --quiet

# Output:
#   project_name/loss_summ_data.tsv          per-gene intactness call
#   project_name/orthology_classification.tsv  one-to-one / one-to-many / many-to-many

TOGA emits eight classes per gene in loss_summ_data.tsv: I (intact), PI (partial intact), UL (uncertain loss), L (lost), M (missing under assembly gap), PM (partial missing), PG (paralogous projection / no orthologous chain found), N (no data). For evolutionary analysis, treat I as functional; PI as fragmented (often functional but caveat); L + UL as candidates for true gene loss after manual review of read coverage; M / PM are assembly-quality issues, not biology; PG indicates the algorithm found only paralogous chains. CESAR 2.0 provides exon-aware coding annotation projection used internally.

Functional Annotation Transfer with eggNOG-mapper

Goal: Propagate GO / KEGG / EC annotations from curated orthologs to a novel proteome.

Approach: Run eggNOG-mapper with appropriate evolutionary level -> integrate with OrthoFinder HOGs for confidence stratification.

emapper.py \
    --output project \
    -i novel_proteome.faa \
    --cpu 16 \
    --decorate_gff genome.gff \
    --tax_scope auto \
    --target_orthologs all \
    --evidence_type experimental \
    --pfam_realign denovo

Output project.emapper.annotations includes per-gene: best ortholog, taxonomic scope of OG, GO terms, KEGG pathways, COG category. Confidence stratification: experimental-evidence GO (EXP, IDA, IPI, IMP, IGI, IEP) > non-experimental (ISO from 1:1 close ortholog) > IEA (electronic). Tag annotation transfer evidence in output.

Reconciliation: When Methods Disagree

Operational rule for publication: Cross-validate single-copy orthologs across 2+ methods (OrthoFinder3 + SonicParanoid2 or OrthoFinder3 + OMA); use consensus HOG set for phylogenomics. Functional annotation transfer requires either 1:1 orthology + low branch dN/dS, or eggNOG ISO/EXP evidence. Report annotation pipeline normalization in methods.

Cohort Gotchas

• WGD lineages: Salmonids (recent Ss4R WGD), teleosts (Ts3R), plants (1-4 rounds), yeast (2 rounds), opisthokonts (1-2 ancient) -- require synteny-aware orthology
• Highly fragmented assemblies: N50 < 100 kb produces extensive false absence; flag affected species and exclude from CAFE
• Polyploids: modern polyploids are multi-genome individuals; assign subgenomes before orthology (see [[whole-genome-duplication]])
• Genome size variation: Drosophila ~150 Mb vs Locust ~5 Gb -- repeat-dominated large genomes have inflated false positive rates from TE-derived proteins; filter TE proteins before orthology
• Domain rearrangements: chimeric gene-fusion proteins (e.g. Jumonji-domain) place into multiple OGs; OrthoFinder3 + Broccoli chimera detection helpful
• HGT-affected genes (prokaryotes): standard orthology returns "vertical orthologs" that may not exist; use ALE-aware approaches (see [[gene-tree-species-tree-reconciliation]])

Anticipated Reviewer Pushback

Common Errors

Tool Installation Notes

# OrthoFinder3
conda install -c bioconda orthofinder=3.0
# Or via Python wrapper: pip install orthofinder3

# SonicParanoid2
conda install -c bioconda sonicparanoid

# Broccoli
git clone https://github.com/rderelle/Broccoli && cd Broccoli && pip install .

# ProteinOrtho 6
conda install -c bioconda proteinortho

# OMA standalone (Linux)
wget https://omabrowser.org/standalone/OMA.tgz && tar xf OMA.tgz && cd OMA && ./install.sh
# FastOMA
pip install fastoma

# eggNOG-mapper 2
conda install -c bioconda eggnog-mapper

# JustOrthologs
git clone https://github.com/ridgelab/justOrthologs && cd justOrthologs && python setup.py install

# TOGA
conda env create -f https://raw.githubusercontent.com/hillerlab/TOGA/master/toga_env.yml
# Requires nf-core and Nextflow

# Quality control
conda install -c bioconda busco compleasm

For Quest-for-Orthologs benchmark submission, follow https://orthology.benchmarkservice.org/ instructions; the benchmark refreshes annually.

References

• Fitch WM 1970 Syst Zool 19:99 (ortholog / paralog definitions)
• Sonnhammer EL & Koonin EV 2002 Trends Genet 18:619 (in-paralog / out-paralog)
• Altenhoff AM & Dessimoz C 2009 PLoS Comp Biol 5:e1000262 (HOG framework)
• Altenhoff AM et al 2012 PLoS Comp Biol 8:e1002514 (ortholog conjecture quantification)
• Altenhoff AM et al 2013 PLoS Comp Biol 9:e1002954 (HOGs at every taxonomic level)
• Altenhoff AM et al 2019 NAR 47:D424 (OMA orthology database)
• Altenhoff AM et al 2024 Genome Biol 25:115 (Quest for Orthologs benchmark)
• Emms DM & Kelly S 2019 Genome Biol 20:238 (OrthoFinder 2)
• Emms DM & Kelly S 2017 MBE 34:3267 (STRIDE rooting)
• Emms DM & Kelly S 2025 bioRxiv 2025.07.15.664860 (OrthoFinder 3)
• Cosentino S & Iwasaki W 2023 NAR 51:e85 (SonicParanoid2)
• Derelle R et al 2020 MBE 37:3389 (Broccoli)
• Lechner M et al 2011 BMC Bioinf 12:124 (ProteinOrtho)
• Majidian S et al 2025 NAR 53:D421 (FastOMA)
• Cantalapiedra CP et al 2021 MBE 38:5825 (eggNOG-mapper 2)
• Miller JB et al 2019 Bioinformatics 35:546 (JustOrthologs 2)
• Kirilenko BM et al 2023 Science 380:eabn3107 (TOGA + CESAR)
• Sharma V & Hiller M 2017 NAR 45:8369 (CESAR 2.0)
• Stamboulian M et al 2020 Bioinformatics 36:i219 (ortholog conjecture revisited)
• Force A et al 1999 Genetics 151:1531 (subfunctionalization)
• Freeling M 2007 PNAS 104:8723 (gene balance hypothesis)
• Nehrt NL et al 2011 PLoS Comp Biol 7:e1002073 (ortholog conjecture challenge)
• Studer RA & Robinson-Rechavi M 2009 Trends Genet 25:210 (ortholog conjecture critique)
• Penel S et al 2009 BMC Bioinf 10:S3 (HOGENOM)
• Zhang C et al 2022 Bioinformatics 38:i131 (ASTRAL-Pro2)
• Di Franco A et al 2019 BMC Eco Evo 19:21 (alignment filtering improves trees)

Related Skills

• comparative-genomics/synteny-analysis - Synteny-anchored ortholog disambiguation in WGD lineages
• comparative-genomics/whole-genome-duplication - Distinguishing ohnologs (WGD paralogs) from orthologs
• comparative-genomics/gene-family-evolution - Birth-death modeling on OG counts requires consistent orthology
• comparative-genomics/gene-tree-species-tree-reconciliation - DTL-aware orthology for prokaryotes / HGT-affected lineages
• comparative-genomics/positive-selection - Selection analysis on single-copy ortholog alignments
• comparative-genomics/comparative-annotation-projection - TOGA / CESAR projects orthology with gene-loss classification
• phylogenetics/modern-tree-inference - Single-copy concatenation phylogenomics input
• phylogenetics/species-trees - Coalescent species-tree from OrthoFinder gene trees
• alignment/multiple-alignment - MSA quality affects OrthoFinder MSA-mode HOG output
• genome-annotation/functional-annotation - Functional annotation propagation via eggNOG / orthology
• read-qc/quality-reports - BUSCO / Compleasm completeness affects orthology reliability