feat: expanded benchmark (95 AMPs + 96 decoys, AUROC 0.7832) + DKP risk + stability calibration

.gitignore

@@ -159,3 +159,4 @@ high_risk_review/*
 tmp/
 temp/
 *.tmp
+.commandcode/

docs/ASSAY_PREREGISTRATION.md

@@ -58,8 +58,11 @@ non-reporting of negative results.
 Priority order: if budget-constrained, run organisms 1, 2, and 3 (minimum viable screen + MDR
 impact). MRSA USA300 is priority 3 because SEED-007 (bombolitin-II), SEED-008 (Trp-rich
 puroindoline-a), and SEED-009 (proline-rich Bac2A) have mechanisms that operate well against
-Gram-positive; MRSA activity is the single highest-impact result achievable with this panel.
-Cost: +$200–400 per peptide at most contract labs.
+Gram-positive. SEED-006 (mastoparan-X, wasp-venom helix insertion) operates against both Gram-
+negative and Gram-positive membranes and provides Gram-negative coverage even in the MRSA-only
+panel. SEED-001_VAR_064 serves as the positive control (magainin-1 derivative, known active).
+MRSA activity is the single highest-impact result achievable with this panel. Cost: +$200–400
+per peptide at most contract labs.
 
 **Note for SEED-009 (proline-rich, top 4 ensemble-score ranks in pilot panel):** Proline-rich
 cathelicidins can show reduced activity in standard Mueller-Hinton Broth — media composition
@@ -140,7 +143,11 @@ TI = HC50 (µg/mL) / MIC (µg/mL)
 > - SEED-003 (11 AA, <15 AA calibration edge; model scores 0.35–0.38 likely underestimate actual t½)
 > - SEED-007 (11 AA; same length as SEED-003; Met at position 6 in all 4 pilot variants — HPLC
 >   purity mandatory at receipt; Nle substitution preferred for oxidation-resistant synthesis)
-> - SEED-008 (13 AA, model calibrated for 18–30 AA; Trp steric effect expected to improve t½)
+> - SEED-008 (13 AA, model calibrated for 18–30 AA; Trp steric effect expected to improve t½).
+>   **ALL 4 SEED-008 pilots carry F-Pro N-terminus: Nα-acetylation is REQUIRED on the synthesis
+>   order** to prevent diketopiperazine (DKP) cyclization (cyclo(F-Pro), MW≈244 Da, t½ hours–days
+>   at 37°C pH 7.4). Confirm full-length peptide by MS at receipt; MW−244 Da satellite peak
+>   indicates DKP contamination — reject batch if satellite exceeds 5% relative peak area.
 > - SEED-009 (Pro-X protease resistance not captured by model; expected to exceed model score)
 > - SEED-005 (safety score 0.845 — lowest in pilot panel; hemolysis assay at MIC/3 mandatory;
 >   if HC₅₀ < 10× MIC, exclude from Wave 2)

docs/DISCOVERY_PREDICTION.md

@@ -482,8 +482,8 @@ Despite the probability gap, the pipeline has done the following correctly:
 2. **Consistent synthesis feasibility:** All pilot candidates are MODERATE SPPS difficulty
    except one HIGH (SEED-002 23-mer). Zero synthesis-impossible candidates nominated.
 
-3. **Strong AUROC:** 0.8420 (bootstrap CI₉₅: 0.76–0.91, n=87, n_bootstrap=2000; PR #72 face_segregation_bonus) — the ensemble correctly separates
-   AMP-like from random-sequence background ~84% of the time.
+3. **Strong AUROC:** 0.7832 (expanded 95-AMP + 96-decoy benchmark, CI₉₅: 0.72–0.84, n=191, n_bootstrap=2000; original demo set 0.8420 on 43+44; PR #110) — the ensemble correctly separates
+   AMP-like from random-sequence background ~78% of the time.
 
 4. **Dual-scorer consensus:** All phase3 nominees have disagreement ≤ 0.45 (physicochemical and
    Boman scorers agree within the validated threshold). Note: SEED-008 (Trp-rich puroindoline-a)

docs/EXPERT_REVIEW_PACK.md

@@ -1,7 +1,7 @@
 # OpenAMP Foundry — Expert Review Pack
 
 **Phase 3 Pilot Panel — Wave 1 Candidates**
-**Pipeline version:** 0.5.x (AUROC 0.8420 on 43-AMP / 44-decoy benchmark)
+**Pipeline version:** 0.5.x (AUROC 0.7832 on expanded 95-AMP / 96-decoy benchmark; original demo set 0.8420 on 43+44)
 **Generated:** 2026-06-28 (updated from 2026-06-27 original)
 **Batch ID:** See `outputs/phase3_manifest.json`
 **Candidate selection manifest SHA-256:** `outputs/phase3_manifest.json`
@@ -30,7 +30,7 @@ biological predictors. No antimicrobial activity has been demonstrated in vitro
 Do not describe any candidate as an antibiotic, drug, cure, therapy, or proven antimicrobial
 without experimental evidence. The lab is the judge.
 
-The pipeline AUROC (0.8420) is a **retrospective benchmark** on the internal 43-AMP reference
+The pipeline AUROC (0.7832) is a **retrospective benchmark** on the expanded 95-AMP / 96-decoy
 set — not an external prospective validation. It indicates the scoring can discriminate known
 AMPs from random peptides on held-out data; it does not predict any individual candidate's
 activity.
@@ -60,7 +60,7 @@ serum stability advantage from protease site model.
 **Pipeline configuration:** `configs/pipeline.yaml` and `configs/phase3.yaml`
 **Selection rule:** `docs/SELECTION_RULE.md` (locked before generation)
 **Full benchmark methodology:** `docs/BENCHMARKING.md`
-**Benchmark AUROC:** 0.8420 (bootstrap CI₉₅: 0.76–0.91, n=87: 43 AMPs + 44 background)
+**Benchmark AUROC:** 0.7832 (bootstrap CI₉₅: 0.72–0.84, n=191: 95 AMPs + 96 background)
 **Pipeline AUPRC:** 0.8627
 
 ---
@@ -211,7 +211,7 @@ Please advise on the following:
 
 ### 6.1 Scientific credibility
 - [ ] Is the physicochemical scoring approach defensible for pre-screening short AMPs (11–15 AA)?
-- [ ] Is the AUROC (0.8420 on 43+44, CI₉₅: 0.76–0.91) sufficient to justify synthesis investment?
+- [ ] Is the AUROC (0.7832 on expanded 95+96 benchmark, CI₉₅: 0.72–0.84) sufficient to justify synthesis investment?
 - [ ] Do you agree that SEED-008 (Trp-rich, non-helical) warrants a different calibration than helical AMPs?
 - [ ] Does the face segregation bonus (helix-wheel moment analysis) reflect a mechanistically sound distinction?
 - [ ] Is the Boman index (2003) a defensible second scorer for the mix of helical and non-helical families?
@@ -271,10 +271,10 @@ The expert reviewer must be aware of these limitations:
 | Limitation | Impact |
 |-----------|--------|
 | Level 0–2 evidence only | Scores are physicochemical heuristics, not validated ML predictions |
-| AUROC = 0.8420 on n=87 | CI₉₅: 0.76–0.91; may not generalise to novel AMP classes outside training distribution |
+| AUROC = 0.7832 on n=191 (expanded) | CI₉₅: 0.72–0.84; 52 new AMPs from 12 taxonomic classes now included (defensins, proline-rich, lantibiotics) — more honest estimate but broader AMP classes still challenge the helic-centric scorer |
 | Safety model blind spot | Melittin scores Safety=1.0; curvature-mediated hemolysis not captured; hemolysis assay mandatory |
 | SEED-008 high disagreement (0.41–0.44) | Boman index penalises Trp; activity_likeness more appropriate for Trp-rich sequences |
-| Serum stability model calibrated for 18–30 AA | SEED-003 (11 AA) and SEED-008 (13 AA) scores may underestimate actual stability |
+| Serum stability model calibrated for 18–30 AA | SEED-003 (11 AA), SEED-007 (11 AA), and SEED-008 (13 AA) scores may underestimate actual stability (short-peptide steric/flexibility effects) |
 | Pro-rich serum stability not in model | SEED-009 model scores (0.572) expected to underestimate actual stability; Pro-X bond resistance is mechanistic |
 | SEED-007 Met oxidation (all 4 pilot variants) | Met at position 6 in all SEED-007 pilot sequences; oxidised Met loses amphipathicity; Nle substitution or inert atmosphere storage required |
 | SEED-009_VAR_033 Met oxidation | VAR_033 (RRLPRPGYMPRP) has Met at position 9 — unique among SEED-009 variants; carries both MET×1 and PROLINE_RICH_INTRACELLULAR QC flags simultaneously; Nle substitution + HPLC purity mandatory at receipt |

docs/ROADMAP.md

@@ -110,7 +110,7 @@ review (2026-06-28). Progress on these would materially raise breakthrough proba
 
 | Gap | Why it matters | Effort estimate |
 |-----|----------------|-----------------|
-| Large-scale benchmark (≥ 500 AMPs vs composition-matched decoys, cluster-split) | Current AUROC 0.8420 measured on 43+44 demo set (n=87, CI₉₅: 0.76–0.91); may not generalise | Medium |
+| ~~Large-scale benchmark (≥ 500 AMPs vs composition-matched decoys, cluster-split)~~ | ~~Current AUROC 0.8420 measured on 43+44 demo set (n=87, CI₉₅: 0.76–0.91); may not generalise~~ | **Partial** (PR #110: expanded to 95 AMPs + 96 decoys, n=191, AUROC=0.7832, CI₉₅: 0.72–0.84; 52 new public-domain AMPs from 12 taxonomic classes; covers defensins, proline-rich, lantibiotics — a more honest estimate. 500+ target still deferred to v1.0+) |
 | External predictor ensemble adapters (CAMPR4, AMPScanner, dbAMP, AntiCP2, Macrel) | Independent second opinions on activity; required for scientific credibility. Manual web-submission checklist at `outputs/external_predict_checklist.md`. Macrel v1.6.0 CLI ONNX bug documented PR #77 — all sequences (incl. canonical AMPs magainin-2, LL-37) misclassified as NAMP; use web server at big-data-biology.org/software/macrel. AMPlify omitted: GPU/ONNX env incompatible with current deps | Medium (checklist generated; web submissions pending) |
 | ~~True novelty check against APD3, DRAMP v3.0, dbAMP~~ | ~~Current novelty scored against 45-sequence seed set only; may overestimate novelty~~ | **Partial** (PR #86: `novelty-check-broad` compares against 72-AMP curated database; result: 16/20 NOVEL, 3 KNOWN_VARIANT, 1 CLOSE_RELATIVE; full APD3 BLASTp still needed before publication) |
 | ~~AUPRC alongside AUROC~~ | ~~Better metric for class-imbalanced AMP datasets~~ | **Done** (PR #58; updated PR #72) — pipeline AUPRC = 0.8627 |

docs/WET_LAB_PROBABILITY.md

@@ -2,11 +2,11 @@
 
 > **Scope**: Quantified probability that the 20-candidate pilot panel will produce ≥1 novel AMP
 > family passing multi-step wet-lab validation and generating a publishable breakthrough finding.
-> Generated: 2026-06-28. Updated 2026-06-28 (PR #86). Reviewed against `outputs/pilot_panel.csv`
-> (7 scaffold families, 20 candidates, pipeline AUROC = 0.8420, bootstrap CI₉₅: 0.76–0.91).
-> See `docs/NOVELTY_BROAD_CHECK.md` for broad novelty analysis (72-AMP reference database);
-> 16/20 candidates confirmed NOVEL, 3 KNOWN_VARIANT (SEED-003, SEED-001 positive control),
-> 1 CLOSE_RELATIVE (SEED-005).
+> Generated: 2026-06-28. Updated 2026-06-29 (expanded benchmark PR #110). Reviewed against `outputs/pilot_panel.csv`
+> (7 scaffold families, 20 candidates, pipeline AUROC = 0.7832, bootstrap CI₉₅: 0.72–0.84,
+> expanded 95-AMP + 96-decoy benchmark, n=191). See `docs/NOVELTY_BROAD_CHECK.md` for broad
+> novelty analysis (72-AMP reference database); 16/20 candidates confirmed NOVEL, 3 KNOWN_VARIANT
+> (SEED-003, SEED-001 positive control), 1 CLOSE_RELATIVE (SEED-005).
 
 ---
 

examples/validation/known_amps.csv

@@ -42,3 +42,55 @@ REF-PLA-001,KKVVFKVKFK,short_cationic,Lee_2004,1
 REF-GKA-001,GKAFVGEIMNS,magainin_c_fragment,Westerhoff_1989,1
 REF-WKL-001,WKLFKKIGAVLKVL,magainin_analog,Dathe_1997,1
 REF-KFL-001,KFLHSAKKFGKAFV,magainin_core,Maloy_1995,1
+REF-LL37-002,LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES,cathelicidin,Gudmundsson_1996_PNAS,1
+REF-HBD1-001,DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK,beta_defensin,Bensch_1995_FEBS_Lett,1
+REF-HBD2-001,GIGDPVTCLKSGAICHPVFCPRRYKQIGTCGLPGTKCCKKP,beta_defensin,Harder_1997_Nature,1
+REF-HBD3-001,GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK,beta_defensin,Harder_2001_JBC,1
+REF-HIS5-001,DSHAKRHHGYKRKFHEKHHSHRGY,histatin,Oppenheim_1988_JBC,1
+REF-DCD1-001,SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL,dermcidin,Schittek_2001_Nat_Immunol,1
+REF-HPCN-001,DTHFPICIFCCGCCHRSKCGMCCKT,hepcidin,Park_2001_JBC,1
+REF-GAE1-001,SMLSVLKNLGKVGLGFVACKINKIQ,gaegurin,Park_1994_BBRC,1
+REF-RNX-001,FLGGLIKIVPAMICAVTKKC,ranalexin,Clark_1994_JBC,1
+REF-JAP1-001,FFPIGVFDKASKVFPTIGV,japonicin,Isaacson_2002,1
+REF-BRV1-001,FLPVLAGIAAKVVPALFCKITKKC,brevinin_1,Morikawa_1992,1
+REF-PHYL-001,FLSLIPHAINAVSAIAKHS,phylloseptin,Leite_2005,1
+REF-DRS1-001,ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ,dermaseptin_S1,Mor_1991,1
+REF-DRS3-001,ALWKNMLKGIGKLAGQAALGAVKTLVGAETQ,dermaseptin_S3,Mor_1994_BIOCHEM,1
+REF-NGR1-001,GLLSGILGAGKHIQCKIRTC,nigrocin,Park_2001,1
+REF-API1-001,GNNRPVYIPQPRPPHPRI,apidaecin_Ia,Casteels_1989_EMBO_J,1
+REF-APIB-001,GNNRPVYIPQPRPPHPRL,apidaecin_Ib,Casteels_1989_EMBO_J,1
+REF-DROS-001,GKPRPYSPRPTSHPRPIRV,drosocin,Bulet_1993_JBC,1
+REF-FORM-001,GRPNPVNNKPTPYPHL,formaecin,Vegh_2002,1
+REF-TN1-001,LCNLERCVYGGCDSGTYSGKCTCTEGGFF,tenecin_1,Moon_1994,1
+REF-DEFA-001,ATCDLLSGTGINHSACAAHCLLRGNRGGYCNGKGVCVCR,defensin_A,Dimarcq_1998,1
+REF-THAN-001,GSKKPVPIIYCNRRSGKCQRM,thanatin,Fehlbaum_1996_PNAS,1
+REF-BMAP-001,GRFKRFRKKFKKLFKKLSPVIPLLHL,BMAP_27,Skerlavaj_1996_FEBS_Lett,1
+REF-BMAP28-001,GGLRSLGRKILRAWKKYGPIIVPIIRI,BMAP_28,Skerlavaj_1996_FEBS_Lett,1
+REF-SMAP-001,RGLRRLGRKIAHGVKKYGPTVLRIIRI,SMAP_29,Brogden_2006,1
+REF-PMAP23-001,RIIDLLWRVRRPQKPKFVTVWV,PMAP_23,Zanetti_1994_FEBS_Lett,1
+REF-PMAP36-001,GRFKRFRKKFKKLFKKLSPVIPLLHLG,PMAP_36,Scocchi_2012,1
+REF-OBAC5-001,RFRPPIRRLPPPRFPIRRLFNPGQDI,OaBac_5,Shamova_1999,1
+REF-OBAC11-001,RLKELFKHPELKTLPKLLKRELEKTLK,OaBac_11,Anderson_2004,1
+REF-BAC7-001,RLCRIVVIRVCR,bactenecin_7,Wu_1999,1
+REF-PRG1-001,RGGRLCYCRRRFCVCVGR,protegrin_1,Kokryakov_1993_FEBS_Lett,1
+REF-PRG2-001,RGGRLCYCRRRFCICVGR,protegrin_2,Kokryakov_1993_FEBS_Lett,1
+REF-PRG3-001,RGGGLCYCRRRFCVCVGR,protegrin_3,Kokryakov_1993_FEBS_Lett,1
+REF-PR39-001,RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRLPPRFPGKR,PR_39,Agerberth_1991,1
+REF-RSA1-001,QCQNNEACKKAGKVEKACCLKFDKTCSGACGSGN,RsAFP_1,Terras_1992,1
+REF-RSA2-001,QCQNNECKKSGKLEKACCEKFDKTCSGACGSGF,RsAFP_2,Terras_1992,1
+REF-ACE1-001,DKCERSSHLLKGCKMTNFLANIFSNLSFDPKTCEGAVPGC,Ace_AMP1,Tassin_1998,1
+REF-SNAK-001,NAFTCMKCTKKPCKKCVFCKYPFEYYY,snakin_1,Segura_1999,1
+REF-PLEC-001,GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCY,plectasin,Mygind_2005_Nature,1
+REF-EURC-001,GFCVNSPWDDEDLKCHNHCKSINGYKGGYCTKGKAVCKCY,eurocin,Schneider_2010,1
+REF-PLUR-001,GWGSFFKKAAHVGKHVGKAALTHYL,pleurocidin,Cole_1997_JBC,1
+REF-PISC1-001,FFHHIFRGIVHVGKTIHLRLTG,piscidin_1,Silphaduang_2001,1
+REF-PISC3-001,FFHHIFRGLIHVGLTIHLRITG,piscidin_3,Silphaduang_2001,1
+REF-MOR1-001,FFRHLFRGSFLVGTLIHLRIG,moronecidin,Lauth_2002_JBC,1
+REF-AREN1-001,RWCVYAYVRVRGVLVRYRRCW,arenicin_1,Ovchinnikova_2004,1
+REF-TACH1-001,KWCFRVCYRGICYRRCR,tachyplesin_I,Nakamura_1988_JBC,1
+REF-TACH2-001,RWCFRVCYRGICYRKCR,tachyplesin_II,Miyata_1989,1
+REF-POLY1-001,RRWCFRVCYRGFCYRKCR,polyphemusin_I,Miyata_1989,1
+REF-TMPG-001,ILSKIFSLLKKVLP,temporin_G,Simmaco_1996_Eur_J_Biochem,1
+REF-RUGA-001,GFLSIVGALSHALPSLISQIK,rugosin_A,Suzuki_2007,1
+REF-NISA-001,ITSISLCTPGCKTGALMGCNMKTATCHCSIHVSK,nisin_A,Gross_1971_JACS,1
+REF-BRV2-001,GLLDSLKGFAATVAKGVAAQVVDKLLKCKLTGKC,brevinin_2,Conlon_2004,1

examples/validation/random_background.csv

@@ -43,3 +43,55 @@ BG-041,NVFSFEAARG,background,uniprot_freq_rng43,0
 BG-042,LPVTKPVVKFT,background,uniprot_freq_rng43,0
 BG-043,TLLVQEVLLGALSH,background,uniprot_freq_rng43,0
 BG-044,RAATKSSALNQNLE,background,uniprot_freq_rng43,0
+BG-045,TGIMDEGTDSNPIKGGFPKLGAATKVVYW,background,uniprot_freq_rng1729,0
+BG-046,LSESAEVSTLGHAGYNELRHNGTLHDRP,background,uniprot_freq_rng1729,0
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+BG-048,EQIQHIVPDSE,background,uniprot_freq_rng1729,0
+BG-049,DQQLTTSKVEFFTTTTFFAVVYRGPSCLKM,background,uniprot_freq_rng1729,0
+BG-050,YAIAAPQGKSMRPVLNPGNAQGESLC,background,uniprot_freq_rng1729,0
+BG-051,YLIGGLVFSVEGDYCVTYGGDMIAP,background,uniprot_freq_rng1729,0
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+BG-053,YKSENSWETNDQRAGLKHAIPSARNLVTITVYRV,background,uniprot_freq_rng1729,0
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