Bio Comparative Genomics Whole Genome Duplication

Version Compatibility

Reference examples tested with: wgd v2.0.31+ (heche-psb/wgd; Chen et al 2024 Bioinformatics 40:btae272), KsRates 1.1.3+ (VIB-PSB/ksrates; Sensalari 2022 Bioinformatics 38:530), DupGen_finder (Qiao 2019 Genome Biol 20:38), MAPS 1.0 (Li 2018), POInT (Conant lab), SLEDGe (bioRxiv 2024.01.17.574559), Whale.jl 2.0+, ksrates pip 1.1+, MCScanX 1.0+, PAML 4.10+ (yn00/codeml for Ks), BLAT 36+, DIAMOND 2.1+, R 4.4+, mclust 6.1+ (for mixture models). Python 3.10+ required for wgd v2.

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

• CLI: wgd --version; ksrates --version; wgd ksd --help
• Python: pip show wgd ksrates
• R: packageVersion('mclust')

If code throws wgd ksd: cannot find PAML output, KsRates: insufficient sister species, MAPS: missing tree, these tools have specific input expectations: wgd needs codon-aware MAFFT/MUSCLE alignment; KsRates needs configured config_ksrates.txt; MAPS needs nucleotide tree. The deprecated arzwa/wgd v1 is replaced by heche-psb/wgd v2.

Whole Genome Duplication Analysis

"Are there WGD events in this lineage and when did they occur?" -> WGD detection combines Ks distributions (synonymous-substitution rates between gene paralog pairs, showing peaks at past polyploidy events) and synteny block analysis (parallel collinear blocks within a genome). Modern best practice uses wgd v2 (Chen et al 2024 Bioinformatics 40:btae272) as an integrated pipeline. KsRates (Sensalari 2022 Bioinformatics 38:530) is mandatory for cross-lineage comparison because substitution rates vary across the tree -- ignoring this places WGDs incorrectly relative to speciation events. The fundamental tradeoff: Ks plot peaks are visually obvious but biologically ambiguous between (1) small-scale tandem duplications, (2) segmental duplications, and (3) true WGD; combining Ks with synteny anchors disambiguates these.

• CLI: wgd dmd and wgd ksd -- paranome construction + Ks distribution
• CLI: wgd syn -- synteny-anchored WGD signal extraction
• CLI: ksrates init then ksrates wgd-paralogs ortho -- substitution-rate-corrected positioning
• CLI: MCScanX -h then dupgen_finder -- duplication-class assignment
• CLI: mapsR -- gene-tree-based WGD phylogenetic placement

Algorithmic Taxonomy

Methodology evolves; verify the current wgd v2 manual and the Chen & Zwaenepoel 2023 review chapter (in Polyploidy: Methods and Protocols) before locking on a single workflow. The 2R vertebrate / 3R teleost / Ss4R salmonid WGDs are well-established; novel WGD claims require concordance across Ks, synteny, and phylogenomic placement.

Decision Tree by Experimental Scenario

Per-Tool Failure Modes

Saturation at Ks > 2

Trigger: Computing Ks for ancient WGD candidates from distantly related taxa.

Mechanism: Synonymous substitutions saturate after Ks ~2; each site has undergone multiple substitutions. Observed Ks underestimates true Ks; the relationship between Ks and time becomes non-monotonic above 1.5. WGD peaks at age 200+ Myr in vertebrates / 100+ Myr in plants are at Ks > 2 and unreliable (Vanneste 2013 MBE 30:177).

Symptom: wgd ksd output shows peak at Ks > 1.5 with broad distribution; KsRates rate-corrected Ks even more uncertain; visual fitting yields ambiguous components.

Fix: Restrict Ks-based WGD inference to Ks < 1.5; for older WGDs, use phylogenomic methods (MAPS, POInT, Whale.jl) which don't rely on Ks alone. Report Ks-based dating with explicit saturation caveat. For 2R vertebrate WGD (~500 Myr), MAPS / Whale.jl is required because Ks is saturated.

Substitution-rate variation across lineages

Trigger: Comparing WGD position in two lineages with different molecular evolutionary rates.

Mechanism: If lineage A evolves twice as fast as lineage B, the same biological time is at twice the Ks in lineage A. A WGD at Ks = 0.5 in lineage A and Ks = 0.25 in lineage B might be the same event biologically.

Symptom: WGD claimed at "different times" in different lineages; speciation-vs-WGD relative timing flips depending on which lineage is the focal species.

Fix: Always use KsRates (Sensalari 2022) when comparing WGDs across lineages. KsRates uses outgroup-species speciation events to calibrate the relative substitution rates, then rescales Ks to a common scale. Single-lineage Ks dating is unreliable for inter-lineage comparison.

Tandem duplication masquerading as WGD peak

Trigger: Ks distribution shows a peak at low Ks (< 0.5); user concludes "recent WGD."

Mechanism: Tandem duplications create paralog pairs with low Ks; these accumulate in clusters (especially in NLR genes in plants, OR genes in mammals). The Ks peak reflects clustered tandem origins, not WGD.

Symptom: "WGD" peak Ks-distribution is dominated by paralogs from tandem clusters; synteny analysis shows few cross-chromosome parallel blocks.

Fix: Use DupGen_finder to classify each paralog pair as tandem / proximal / transposed / dispersed / WGD. The true WGD signal comes from the WGD-classified pairs. Recompute Ks distribution restricted to WGD-classified pairs (or anchor-pair restricted to synteny blocks). wgd v2 integrates this filtering.

Synteny block age inconsistency

Trigger: Different synteny blocks from the same suspected WGD have different Ks distributions.

Mechanism: Real WGD blocks should share a synchronized Ks distribution centered at the WGD age; if blocks have very different ages, the inferred "WGD" was probably a series of segmental duplications.

Symptom: Per-block Ks median varies widely; some blocks show Ks ~ 0.3 and others Ks ~ 1.0; not consistent with single WGD.

Fix: Compute per-block Ks distribution; require synchronization (interquartile range overlapping across blocks). wgd v2 reports per-block age dispersion. If dispersion is high, downgrade to "potentially WGD-like" or attribute to ancient segmental duplication history.

Mixture model under/overfitting components

Trigger: Fitting GMM (gaussian mixture model) or ELMM (exponential-lognormal mixture model) to Ks distribution.

Mechanism: Mixture models with too few components miss real WGD signal; too many components find spurious peaks. BIC-based model selection is standard but sensitive to bin choice in histograms.

Symptom: Different runs with different bin counts give different component numbers; visual peaks don't match component means.

Fix: Use BIC-based component selection with 5-fold cross-validation; report uncertainty in component count. wgd v2 fits both GMM and ELMM and reports BIC for each model up to 5 components. Visually validate against synteny block analysis.

Comparing wgd v1 (deprecated) vs v2 outputs

Trigger: Mixing scripts written for arzwa/wgd v1 with heche-psb/wgd v2.

Mechanism: wgd v1 was deprecated 2023; v2 has different command-line interface, default parameters, and output file structure. Scripts from v1 fail or produce different results in v2.

Symptom: Older scripts using wgd ksd v1 syntax don't work in v2; output paths differ.

Fix: Update all scripts to wgd v2 syntax. The v1 wgd is no longer maintained; v2 is the current standard.

KsRates failing with insufficient species

Trigger: Running KsRates with one focal species and one outgroup.

Mechanism: KsRates calibrates substitution-rate variation via at least two outgroup speciation events; one outgroup species provides only one rate point and cannot resolve rate heterogeneity.

Symptom: KsRates errors or produces unreliable rate-corrected Ks.

Fix: Include >= 2 outgroup species at different phylogenetic distances; for plants, a model angiosperm + gymnosperm pair; for vertebrates, lamprey + invertebrate outgroup. Document outgroup sampling.

Polyploid genome confusion

Trigger: Running wgd v2 on an unassigned-subgenome polyploid (e.g. wheat hexaploid without A/B/D subgenome labels).

Mechanism: WGD signal is muddled when subgenomes are not separated; orthologs across subgenomes appear as paralogs, intermixing tandem / segmental / WGD / homeolog signal.

Symptom: Ks distribution shows multiple overlapping peaks at varying intensities; component fits are unstable.

Fix: Assign subgenomes first using k-mer methods (KMC2 + SubPhaser; Jia 2022 New Phytol 235:801) or synteny-based assignment (GENESPACE; Lovell 2022). Then run wgd v2 on each subgenome separately. Document subgenome assignment.

Confusion of homeologous (WGD) vs orthologous (speciation) pairs

Trigger: Computing Ks across recent WGD species without explicit homeolog labeling.

Mechanism: In recent WGD lineages (e.g. salmonid Ss4R), the "ortholog" between species and the "homeolog within species" can have similar Ks; standard ortholog detection (OrthoFinder) lumps both.

Symptom: wgd v2 reports Ks distributions but doesn't distinguish homeologs from orthologs; downstream dating confused.

Fix: Use synteny-anchored ortholog detection (GENESPACE, ProteinOrtho-synteny) to separate cross-species orthologs from within-species homeologs. Run wgd v2 separately on each class. Report each Ks distribution explicitly.