bio-comparative-genomics-positive-selection

## Version Compatibility Reference examples tested with: PAML 4.10.7+, HyPhy 2.5.62+ (BUSTED-MH from Lucaci 2023 MBE 40:msad150; FUBAR-MH from same), datamonkey.org 2024+ for web jobs, IQ-TREE 2.3.6+, MACSE V2.07+, PRANK 170427+, MAFFT 7.526+, PREQUAL 1.02+, HmmCleaner 0.243+, GARD (HyPhy bundled), RDP5 5.59+, ete4 4.1.0+, BioPython 1.84+, scipy 1.13+, polyDFE 2.0+, DFE-alpha 2.16+, GRAPES 1.1.1+, RERconverge 0.3.0+, CSUBST 1.6.0+, PhyloAcc 2.4.0+. Quest-for-Selection benchmark refreshed annually. Before using code patterns, verify installed versions match. If versions differ: - CLI: `codeml` (PAML; check by `codeml /dev/null` -- prints version banner), `hyphy --version`, `gard --help` - Python: `pip show pyhyphy`; introspect ete4 API for tree-labeling - R: `packageVersion('RERconverge')`; `?correlateWithBinaryPhenotype` If code throws `branch-site test LRT non-positive`, `omega2 hit upper bound 999`, `MEME ML mixed gradient`, the most common cause is alignment error or saturated dS -- inspect alignment with TCS / Guidance2 and dS-vs-divergence-time. PAML 4.10 changed several control-file keywords from 4.9 (`getSE = 1` syntax tightened). # Positive Selection Analysis **"Is this gene / branch / site under positive selection?"** -> dN/dS (omega = nonsynonymous-to-synonymous substitution rate ratio) framework with explicit choice of WHICH question is being asked (gene-wide / branch-specific / site-specific / episodic) and WHICH null is being rejected. The "test failed because of selection" claim has more known confounders than any other comparative-genomics inference; **mandatory pre-screens are: recombination (GARD), alignment errors (PREQUAL or HmmCleaner), saturation (dS distribution), and gBGC (W->S substitution bias)**. Skipping any one inflates Type-I error to ~20-50% (Anisimova & Yang 2007 MBE 24:1219; Pond 2006 Mol Biol Evol 23:1891). - CLI: `codeml` PAML site, branch, branch-site models - CLI: `hyphy busted` `hyphy meme` `hyphy fel` `hyphy fubar` `hyphy absrel` `hyphy relax` `hyphy gard` - Web: datamonkey.org for HyPhy jobs without local install - R: `RERconverge::correlateWithBinaryPhenotype()` for trait-rate associations - CLI: `csubst analyze` for convergent substitution - R/CLI: `phyloacc` for noncoding accelerated evolution ## Algorithmic Taxonomy | Method | Question | Null model | Strength | Fails when | |--------|----------|------------|----------|------------| | PAML codeml M0 (Yang 1997 CABIOS 13:555) | Gene-wide single-omega estimate | -- (point estimate) | Standard reference omega; baseline test | Site heterogeneity (use M3+) | | codeml M1a vs M2a (Yang 2000 Genetics 155:431) | Any site under selection? | Nearly neutral, 2-category | Conservative; LRT df=2 | Low power for episodic selection | | codeml M7 vs M8 | More-sensitive site test | Beta(0,1) | Higher power than M1a/M2a | Higher false-positive rate; relaxed-constraint mimics selection | | codeml M8 vs M8a (Swanson 2003 MBE 20:18) | Conservative site test (omega2 = 1 null) | Beta + omega2=1 | Cleanest LRT df=1; preferred site test | Lower power than M7 vs M8 | | codeml branch-site mod A (Zhang 2005 MBE 22:2472) | Selection on pre-specified foreground branch | A1 (omega2=1 fixed) | Most powerful for episodic per-branch selection | Foreground specified post hoc -> Type-I inflation | | codeml clade model (Bielawski & Yang 2004 J Mol Evol 59:121) | Different omega between named clades | M3 with shared categories | Tests for shifted selection regime | Requires clade pre-specification | | codeml free-ratio | Per-branch omega estimates (exploratory) | M0 | Visualizes branch-wise variation | Unidentifiable for short branches; no formal LRT | | HyPhy BUSTED (Murrell 2015 MBE 32:1365) | Any episodic selection on any branch site? | No omega+ class | Site + branch joint; foreground assignable | Sensitive to alignment errors | | HyPhy BUSTED-S (Wisotsky 2020 MBE 37:2430) | BUSTED with synonymous-rate variation | -- | Corrects for SRV; reduces false positives | Slightly less power than BUSTED | | HyPhy BUSTED-MH (Lucaci 2023 MBE 40:msad150) | BUSTED with multi-nucleotide substitutions | -- | Captures complex (multi-hit) substitutions; reduces false positives from MNMs | Newer; limited benchmarking | | HyPhy BUSTED-PH | Two phenotypes; selection on one not other | -- | Tests phenotype-specific selection | Requires phenotype branch label | | HyPhy MEME (Murrell 2012 PLoS Genet 8:e1002764) | Per-site episodic selection | FEL | Detects sites under episodic positive selection | Higher false-positive rate at p threshold | | HyPhy FEL (Kosakovsky Pond 2005 MBE 22:1208) | Per-site pervasive selection | -- | Fast; counts substitutions per site | No episodic detection | | HyPhy FUBAR (Murrell 2013 MBE 30:1196) | Bayesian per-site pervasive selection | -- | Scales to 1000s of sequences; posterior probability | No episodic detection | | HyPhy SLAC | Counting-based fast estimator | -- | Very fast; rough estimate | Lower power; no statistical model | | HyPhy aBSREL (Smith 2015 MBE 32:1342) | Branch-specific selection without pre-specification | -- | Adaptive per-branch omega categories; corrects multiple testing | Multiple-testing burden across many branches | | HyPhy RELAX (Wertheim 2015 MBE 32:820) | Selection relaxation (k<1) or intensification (k>1) | -- | Detects RELAXED selection; cannot be done by other tests | Not designed for adaptive evolution per se | | HyPhy GARD (Pond 2006 MBE 23:1891) | Recombination breakpoint detection | No recombination | MANDATORY pre-screen for any selection test | Computationally heavy; > 50 sequences slow | | McDonald-Kreitman (McDonald & Kreitman 1991 Nature 351:652) | Adaptive substitution rate alpha from poly + div data | Neutral mutation accumulation | Per-gene alpha; population genetics native | Slightly deleterious bias (downward); fixed by asymptotic alpha | | Asymptotic alpha (Messer & Petrov 2013 PNAS 110:8615) | MK with slightly deleterious correction | -- | Unbiased alpha; works at low MAF SFS | Requires SFS data | | impMKT (Murga-Moreno 2022 G3 12:jkac206) | MK with conservative imputation | -- | Gene-level evidence; faster than alpha asymptotic | Less unbiased than asymptotic alpha | | polyDFE (Tataru & Bataillon 2019 Bioinformatics 35:2868) | Full DFE + alpha jointly | -- | Quantifies the distribution of fitness effects | Computational cost; requires polymorphism data | | DFE-alpha (Eyre-Walker & Keightley 2009 MBE 26:2097) | Faster DFE method | -- | Standard DFE inference; many simulated DFEs | Requires demographic correction | | GRAPES (Galtier 2016 PLoS Genet 12:e1005774) | DFE on neutral + selected sites | -- | Joint demography + alpha; robust | Genome-scale dataset required | | RERconverge (Kowalczyk 2019 Bioinformatics 35:4815; Redlich 2024 MBE 41:msae210) | Relative-rate shifts correlated with categorical phenotype | -- | Phylogenome-wide trait associations | Inherits all dN/dS confounders | | CSUBST (Fukushima & Pollock 2023 Nat Eco Evo 7:155) | Convergent substitutions across independent lineages | -- | Combinatorial-substitution omegaC ratio; null-corrected | Requires multi-clade dataset | | PhyloAcc (Hu 2019 MBE 36:1086; Thomas 2024) | Bayesian convergent accelerated noncoding rate | -- | For noncoding elements (CNEs); convergent rate shifts | CDS analyses prefer codon-based methods | | phyloP (Pollard 2010 GR 20:110) | Per-site noncoding rate test | -- | Simple; widely used for noncoding | No convergence; site-by-site | | PRANK + codeml pipeline | Codon-aware MSA + codeml | -- | Standard publication-grade workflow | Slow for large datasets | Methodology evolves; verify the latest HyPhy / PAML manuals and the Álvarez-Carretero "Beginner's Guide" (Álvarez-Carretero et al 2023 MBE 40:msad041) before locking on a single method. The BUSTED-MH and FUBAR-MH (multi-hit) extensions specifically address known Type-I inflation from multi-nucleotide substitutions and are now recommended over basic BUSTED / FUBAR. ## Decision Tree by Experimental Scenario | Scenario | Recommended approach | Why | |----------|------------------------|-----| | Single gene, mammalian (~60 Myr), pre-specified foreground branch | codeml branch-site mod A AND HyPhy aBSREL on foreground | Mutual validation; mod A LRT df=1 + aBSREL adaptive site classes | | Single gene, deep eukaryote (~500+ Myr), no foreground hypothesis | GARD pre-screen -> BUSTED-MH gene-wide -> MEME for sites | Episodic-selection-only methods; saturation-aware (HyPhy under MG94 codon model) | | Genome-wide scan, vertebrates | codeml M7 vs M8 OR HyPhy FUBAR-MH per gene; FDR-correct | Pervasive-selection sites; multi-hit correction critical at scale | | Episodic selection scan | HyPhy MEME genome-wide (per gene); FDR-correct | Site-level episodic detection | | Branch-specific selection on unspecified branches | HyPhy aBSREL | Adaptive per-branch test with built-in multiple-testing | | Comparing selection regimes between two phenotypes | HyPhy BUSTED-PH or RELAX | Phenotype-specific or relaxation-detection | | Recently diverged species (low divergence) | MK / asymptotic alpha (population genetics) | Codon dN/dS unreliable at low divergence; SFS-based instead | | Within-species, dense polymorphism + divergence | polyDFE / GRAPES / asymptotic-MK | Full DFE + alpha jointly; preferred for adaptive-substitution rate | | Coding selection genome-wide, with SFS available | grapes -m AUTO_ALL | Demography-aware alpha; standard population-genetics-aware adaptive-substitution scan | | Noncoding accelerated evolution (CNEs / ECRs) | PhyloAcc, phyloP-acc | Codon-based unsuitable; PhyloAcc Bayesian convergence | | Convergent substitutions across independent lineages | CSUBST | Combinatorial-substitution omegaC; null-corrected | | Trait-correlated rate shifts genome-wide | RERconverge | Categorical / binary phenotype; correlates RERs across thousands of genes | | Suspected positive selection but dS > 2 | Use protein-level method or reduce taxon sampling | Codon-based methods unreliable at saturation; protein-only ASR can still work | | Recombination expected (immune genes, viral genomes) | GARD pre-screen mandatory | Recombination + tree-based selection -> false positives (Anisimova 2003) | | Convergent codon substitution at specific sites | TDG09 (Tamuri 2009) or PCOC (Rey 2018) | TDG09 detects site-specific shifts in selective constraint between trait-defined lineage groups; PCOC detects convergent amino-acid substitution | | Drug-target evolution screen | aBSREL on candidate genes; cross-validate with MEME | Recent positive selection at drug-target loci | | Pathogen / immune-evasion gene with high dS variation | BUSTED-S (synonymous rate variation aware) | dS variation across sites violates basic BUSTED assumptions | | Plasmodium / Trypanosoma / Plasmid analysis | BUSTED-MH (multi-hit aware) | Multi-nucleotide substitutions common in these; basic BUSTED inflates false positives | ## Per-Method Failure Modes ### Recombination producing false positive selection **Trigger:** Running codeml or BUSTED on a gene with recombination breakpoints (viral genes, immune genes, paralog families). **Mechanism:** All single-tree codon models assume one phylogeny across all sites. Recombination produces different trees for different segments; treating them as one tree forces the model to invent rate variation that mimics positive selection (Anisimova et al 2003 Genetics 164:1229). **Symptom:** PAML M8 strongly rejects M7 (LRT > 50), with omega2 = 999 (PAML upper bound) at several "selected sites"; HyPhy BUSTED highly significant; sites clustered in specific gene regions. **Fix:** **MANDATORY: run GARD before any positive selection test.** If GARD detects breakpoints (p < 0.05), partition the alignment at breakpoints and analyze each segment separately, or use the recombination-aware MEME with the partitioned tree set. RDP5 (Martin 2021 Virus Evol 7:veaa087) is an alternative for viral genomes. GARD output `.json` lists breakpoint positions and posterior support. ### Alignment errors producing false positives **Trigger:** Using default MAFFT or MUSCLE alignment on divergent CDS sequences; skipping codon-aware aligner. **Mechanism:** Frame-shifted or misaligned codons introduce apparent non-synonymous substitutions at every position; codon-aware tools see these as positive selection (Schneider 2009 GBE 1:114; Markova-Raina & Petrov 2011 GR 21:863). **Symptom:** "Selected sites" cluster in alignment regions with > 30% gaps; per-site posteriors in BEB / FUBAR concentrate in ambiguous columns; PREQUAL or Guidance2 marks these regions as poorly aligned; protein alignment shows obvious mismatches. **Fix:** Use codon-aware aligner: **PRANK** (Loytynoja 2014 Methods Mol Biol 1079:155) is the standard for selection analysis (correctly models insertions); MACSE V2 (Ranwez 2018 MBE 35:2582) handles frameshifts and pseudogenes natively; OMM_MACSE wrapper combines them. After alignment, filter with PREQUAL (segment-level) or HmmCleaner (Di Franco 2019 BMC Evol Biol 19:21); do NOT use block-filtering (Gblocks, trimAl) which removes informative sites. Segment-level filtering preferred for selection (Di Franco 2019). ### Saturated synonymous sites **Trigger:** Comparing distantly related taxa (deep eukaryotic divergence, > 100 Myr); dS > 3 across most pairs. **Mechanism:** Synonymous sites have undergone multiple substitutions; the observed dS underestimates true dS. The model can't recover the true rate; omega = dN/dS becomes unstable at the upper bound or low (depending on which direction the bias goes). **Symptom:** PAML M0 omega = 999 or near-zero; per-branch dS variance huge; sites with omega > 1 in M8 BEB are at conserved residues (paradox). **Fix:** Reduce taxon sampling to species with dS < 2 on internal branches. For deep selection inference on conserved residues, use protein-level methods (BUSTED with `--model GTR` AA codon translation; aBSREL with protein model option) or restrict to subclade with reasonable saturation. Yang 2007 PAML manual recommends dS < 1.5 per branch. ### gBGC inflating apparent positive selection **Trigger:** Mammalian / vertebrate gene with W->S substitution bias on a fast-evolving lineage. **Mechanism:** GC-biased gene conversion fixes A/T -> G/C alleles preferentially in regions of high recombination, independent of selection (Galtier & Duret 2007 Trends Genet 23:273; Capra 2013 PLoS Genet 9:e1003684). Standard codon models attribute this to positive selection because nonsynonymous substitutions are unequally distributed across codon positions. **Symptom:** Branch with apparent positive selection sits in high-recombination region; W->S / S->W substitution ratio > 1.5; selected sites concentrate at non-degenerate codon positions; HyPhy MEME-MH and BUSTED-MH attribute signal to multi-hit rather than positive selection. **Fix:** Test for gBGC: W->S substitution rates on selected branch / S->W rates; report ratio. Re-run selection analysis with HyPhy BUSTED-MH (multi-hit aware); if signal vanishes, the original "selection" was gBGC + multi-hit substitutions. For genome-wide scans, mask sub-telomeric / high-recombination regions. ### Branch-site test foreground specification **Trigger:** Running codeml branch-site mod A after looking at the data to choose foreground branch. **Mechanism:** The branch-site test is designed for a single a priori foreground; post hoc specification inflates Type-I by ~5x because the choice was informed by the data. **Symptom:** Branch-site test highly significant for the "interesting" branch; aBSREL on same data shows no significant branch (aBSREL has built-in multiple-testing correction). **Fix:** Specify foreground branches in registered protocol before looking at data. For exploratory branch-wise analysis, use aBSREL (Smith 2015 MBE 32:1342) which adaptively assigns branch-specific omega classes with multiple-testing built in. If branch-site test was post hoc, apply Bonferroni correction across all branches tested + report explicitly. ### LRT critical value confusion **Trigger:** Computing branch-site test p-value using standard chi-square df=2. **Mechanism:** The branch-site test compares mod A (4 omega classes) against mod A1 (omega2 fixed at 1). The LRT statistic distribution is a 50:50 mixture of point-mass-at-0 and chi-square(df=1), not chi-square(df=2) (Self & Liang 1987 JASA 82:605; Zhang 2005 MBE 22:2472; Wong 2004 Genetics 168:1041). Using df=2 makes the test conservative; using df=1 standard makes it anticonservative. **Symptom:** Branch-site p-values incorrectly inflated or deflated; users report finding selection at very stringent thresholds. **Fix:** Use the 50:50 mixture critical value: 2.71 at p=0.05 (NOT 3.84). PAML's `chi2 1 LRT` command applies the mixture. Many published applications use chi-square df=2 conservatively, which loses power but doesn't inflate; chi-square df=1 directly is wrong and inflates Type-I. ### omega2 hitting upper bound (999) **Trigger:** PAML codeml output shows omega2 = 999 for an "under selection" site class. **Mechanism:** PAML codeml uses an internal upper bound of 999 (= "infinity" in single precision). Hitting it indicates numerical issue: extremely few synonymous sites in the selected class, dS underestimation, or numerical optimization failure. **Symptom:** Sites flagged as positive selection have omega2 = 999; BEB posteriors for those sites are weirdly distributed. **Fix:** Re-run with multiple starting values of omega (`fix_omega=0`, vary `omega = 0.1, 0.5, 1.0, 2.0, 5.0` across runs); check that all converge to same omega. Inspect alignment at flagged sites for unusual residue conservation. If omega = 999 persists, the gene may have rare-substitution patterns; switch to BUSTED-MH which accounts for multi-hit substitutions. ### Multiple-testing burden in genome scans **Trigger:** Running selection tests across thousands of genes without correction. **Mechanism:** With ~5000 protein-coding genes in a typical analysis, 250 will be significant at p=0.05 under H0. The false-discovery rate without correction is 50%. **Symptom:** Implausibly large gene lists "under selection"; functional categories enriched are non-specific (e.g. all immune genes by FDR). **Fix:** Apply FDR correction (Benjamini-Hochberg). Genes in syntenic regions are non-independent; use Benjamini-Yekutieli for stronger control under dependence. For HyPhy site-level methods, the per-site p < 0.1 default is a starting point; multiple-test correction within a gene is typically not applied (sites within a gene are dependent), but cross-gene correction is necessary. Holm-Bonferroni for strict Type-I. ### Convergent substitution misinterpreted as positive selection **Trigger:** Lineage-specific selection found at a residue that has independently changed in multiple unrelated lineages. **Mechanism:** Convergent substitutions at the same site in independent lineages produce signals in branch-site and other tests; this is convergence, not adaptive evolution per se (though convergent residues often ARE adaptive). **Symptom:** Same residue flagged in multiple unrelated lineages by branch-site test; alignment shows convergent substitutions. **Fix:** Switch from selection test to convergence test: CSUBST (Fukushima & Pollock 2023 Nat Eco Evo 7:155) for combinatorial substitution analysis; RERconverge (Redlich 2024 MBE 41:msae210) for relative-rate-vs-phenotype across categorical traits; PCOC (Rey 2018) for biophysical convergence. Report both convergence test and selection test results. ## Quantitative Thresholds | Quantity | Threshold | Source / Rationale | |----------|-----------|-------------------| | dN/dS interpretation | omega < 1 purifying; omega = 1 neutral; omega > 1 positive (per site, branch, or gene depending on model) | Yang & Bielawski 2000 TREE 15:496; foundational | | Branch-site test LRT critical value | 2.71 at p=0.05 (50:50 mixture chi^2) | Self-Liang 1987 JASA 82:605; Zhang 2005 MBE 22:2472 | | Site-level p-value default | p <= 0.1 (FEL, MEME, FUBAR); FUBAR posterior >= 0.9 | Murrell 2012/2013; Datamonkey conventions | | BEB posterior probability | >= 0.95 significant; >= 0.99 highly significant | Yang & Bielawski 2000 | | dS upper limit for reliability | dS < 1.5 per branch; dS < 3 overall | Yang 2007 PAML manual | | Minimum sequences for codeml | >= 8 with sufficient divergence | Anisimova et al 2001 MBE 18:1585 | | Branch-site test minimum lineages | >= 20 in tree; >= 4 background branches | Yang 2007 | | GARD breakpoint significance | p < 0.05 to partition alignment | Pond 2006; mandatory pre-screen | | MK alpha threshold | alpha > 0 indicates adaptive substitutions; report 95% CI | Smith & Eyre-Walker 2002 | | Asymptotic alpha minimum SFS density | >= 50 sites per frequency bin | Messer & Petrov 2013 |