cschanhniem · Jun 28, 2026 · Jun 28, 2026 · Jun 28, 2026
diff --git a/docs/DISCOVERY_PREDICTION.md b/docs/DISCOVERY_PREDICTION.md
@@ -3,7 +3,7 @@
 **Pipeline:** OpenAMP-Foundry v0.1.0  
 **Date:** 2026-06-28 (updated 2026-06-28)  
 **Status:** Pre-synthesis scientific assessment — for expert review before ordering  
-**Completed improvements:** Serum stability scoring (PR #31/#32), Family diversity cap (PR #31), Reference set expansion 44→73 sequences (PR #33), Net charge pH 7.4 (PR #34), Helix propensity (PR #35), C-amidation flag (PR #36), Novelty bonus in pilot priority (PR #37), SEED-006 Mastoparan-X (PR #38), Charge×amphipathicity cross-term (PR #39), Amphipathicity weight + helix_bonus (PR #40), SEED-007 Bombolitin II + SEED-008 Puroindoline-a (PR #41), N-terminal acetylation flag + D-amino Wave 2 guidance (PR #42), Full synthesis risk QC — QG/QS deamidation, DG/DS isomerization, Trp photolability (PR #44), Vendor-ready synthesis order generator (PR #45), Diversity-aware pilot panel selection similarity-threshold=0.75 (PR #46), Selectivity proxy + HIGH_CYTOTOX_RISK flag — charge/GRAVY-based mammalian cytotoxicity risk detector (PR #47), selectivity_proxy routed into pilot_priority formula — low-cytotox-risk candidates gently demoted (PR #48), Elastase resistance (HNE 3-protease stability model) + aggregation propensity scoring (synthesis feasibility penalty) (PR #49), Aggregation-safe mutation generation + balanced K/R charge variants + SynthQC continuous aggregation score (PR #50), Proline synthesis penalty + helix bonus enhancement — AUROC 0.8138 → 0.8164 (PR #51), Safety pH74 charge consistency + stronger cytotoxicity penalty — SEED-004 net selectivity swing increased 0.035→0.055 (PR #53), AUPRC metric + SEED-009 (Bac2A proline-rich) + SEED-010 (KR-12 LL-37 fragment) (PR #58), ATCC strain corrections + preregistration assay protocol + negative result schema (PR #60), **CRITICAL FIX: SEED-008 Trp-rich scaffold reinstated in synthesis pool (was excluded by Boman W scale artifact); all 10 seeds generated; SEED-007/009 newly included; SEED-004 correctly excluded by safety gate; 6 mechanism-diverse scaffolds confirmed in synthesis pool** (PR #61), Phase3.yaml AUROC documented (0.7936→0.7890 after PR #65) (PR #63), Stale threshold/reference-count docs corrected (PR #64), **Trp-weighted aromatic bonus (1.5× Trp vs Phe/Tyr; Wimley-White interfacial mechanism); safety abs() bug fix (negative charge does not cause hemolysis); Eisenberg scale comment fix; pool SEED-006: 11→10, SEED-009: 18→19; AUROC 0.8164→0.8086** (PR #65)
+**Completed improvements:** Serum stability scoring (PR #31/#32), Family diversity cap (PR #31), Reference set expansion 44→73 sequences (PR #33), Net charge pH 7.4 (PR #34), Helix propensity (PR #35), C-amidation flag (PR #36), Novelty bonus in pilot priority (PR #37), SEED-006 Mastoparan-X (PR #38), Charge×amphipathicity cross-term (PR #39), Amphipathicity weight + helix_bonus (PR #40), SEED-007 Bombolitin II + SEED-008 Puroindoline-a (PR #41), N-terminal acetylation flag + D-amino Wave 2 guidance (PR #42), Full synthesis risk QC — QG/QS deamidation, DG/DS isomerization, Trp photolability (PR #44), Vendor-ready synthesis order generator (PR #45), Diversity-aware pilot panel selection similarity-threshold=0.75 (PR #46), Selectivity proxy + HIGH_CYTOTOX_RISK flag — charge/GRAVY-based mammalian cytotoxicity risk detector (PR #47), selectivity_proxy routed into pilot_priority formula — low-cytotox-risk candidates gently demoted (PR #48), Elastase resistance (HNE 3-protease stability model) + aggregation propensity scoring (synthesis feasibility penalty) (PR #49), Aggregation-safe mutation generation + balanced K/R charge variants + SynthQC continuous aggregation score (PR #50), Proline synthesis penalty + helix bonus enhancement — AUROC 0.8138 → 0.8164 (PR #51), Safety pH74 charge consistency + stronger cytotoxicity penalty — SEED-004 net selectivity swing increased 0.035→0.055 (PR #53), AUPRC metric + SEED-009 (Bac2A proline-rich) + SEED-010 (KR-12 LL-37 fragment) (PR #58), ATCC strain corrections + preregistration assay protocol + negative result schema (PR #60), **CRITICAL FIX: SEED-008 Trp-rich scaffold reinstated in synthesis pool (was excluded by Boman W scale artifact); all 10 seeds generated; SEED-007/009 newly included; SEED-004 correctly excluded by safety gate; 6 mechanism-diverse scaffolds confirmed in synthesis pool** (PR #61), Phase3.yaml AUROC documented (0.7936→0.7890 after PR #65) (PR #63), Stale threshold/reference-count docs corrected (PR #64), **Trp-weighted aromatic bonus (1.5× Trp vs Phe/Tyr; Wimley-White interfacial mechanism); safety abs() bug fix (negative charge does not cause hemolysis); Eisenberg scale comment fix; pool SEED-006: 11→10, SEED-009: 18→19; AUROC 0.8164→0.8086** (PR #65), **Duplicate validation entry removed (REF-GIG-001 = magainin-2 = REF-MAG-001 counted twice); corrected AUROC 0.8086→0.8047 (pipeline), 0.7890→0.7846 (phase3); 43-AMP + 44-background set (n=87)** (PR #66)
 
 ---
 
@@ -243,8 +243,8 @@ Fmoc SPPS with acetonitrile/water gradient; verify MALDI-TOF pre-assay.
 Updated from ~55–65% due to addition of proven scaffold families.
 
 Basis:
-- Pipeline AUROC = 0.8086 (95% CI: 0.71–0.90) vs composition-matched UniProt decoys (PR #65 update; previous 0.8164 after Trp bonus scientific fix; within bootstrap CI)
-- Pipeline AUPRC = 0.8495 (+0.35 above random baseline 0.50; PR curve emphasises precision at selection operating point)
+- Pipeline AUROC = 0.8047 (95% CI: 0.71–0.89) vs composition-matched UniProt decoys (PR #66 dedup fix; 43 unique AMPs, n=87 total; previous 0.8086 pre-dedup, 0.8164 pre-Trp-bonus)
+- Pipeline AUPRC = 0.8443 (+0.2943 above random baseline 0.4943; PR curve emphasises precision at selection operating point)
 - Recall@20 = 43% on internal benchmark (positives recovered in top 20 ranked candidates)
 - SEED-003 family (up to 4/20): RRWQWRMKKLG is curated known AMP → variants ~65–75% hit rate
 - SEED-007 family (up to 4/20): Bombolitin II (*Bombus pennsylvanicus* bumblebee venom) with
@@ -584,7 +584,7 @@ Executing all four actions on the best Wave 1 hits would push the combined proba
 | Stage | Gate | Original | After PRs #31–38 | After PRs #39–42 | After PRs #43–47 | After PRs #48–53 (current) | Primary limiting factor |
 |-------|------|----------|-----------------|-----------------|-----------------|--------------------------|------------------------|
 | 0 | Synthesis success | ~90% | ~90% | ~88% | **~89%** ✓ | **~90%** ✓ (agg model + agg-safe gen + pro penalty + pH74 charge) | SEED-008 W-rich; all aggregation/synthesis risks modelled |
-| 1 | MIC ≤ 32 μg/mL | ~55–65% | ~55–65% | ~60–70% | **~60–70%** | **~61–71%** ✓ (AUROC 0.8086) | AUROC 0.8086 (AUPRC 0.8495); 6 scaffold families confirmed |
+| 1 | MIC ≤ 32 μg/mL | ~55–65% | ~55–65% | ~60–70% | **~60–70%** | **~61–71%** ✓ (AUROC 0.8047) | AUROC 0.8047 (AUPRC 0.8443); 6 scaffold families confirmed |
 | 2 | TI > 10 (selectivity) | ~35–50% | ~35–50% | ~38–52% | **~40–55%** ✓ | **~42–57%** ✓ (stronger SEED-004 demotion) | sel_proxy doubled penalty for HIGH_CYTOTOX_RISK tier |
 | 3 | t½ > 2 h (serum) | ~10–20% | ~25–40% | ~28–42% | **~28–42%** | **~29–44%** ✓ (3-protease model) | Wave 2 D-amino plan machine-readable |
 | 4 | Scaffold novelty | ~10–15% | ~18–25% | ~25–35% | **~26–36%** ✓ | **~26–36%** | Diversity filter removes cross-seed near-dups |
@@ -660,7 +660,7 @@ The corrected estimate is more conservative because:
 
 ### 2. Benchmark Limitations
 
-The AUROC 0.8086 (phase3 synthesis gate: 0.7890) is measured on a small 44+44 demo dataset, not validated against the full
+The AUROC 0.8047 (phase3 synthesis gate: 0.7846) is measured on a small 43+44 demo dataset, not validated against the full
 APD3 (> 3,000 AMPs), DRAMP v3.0 (> 19,000 entries), or ESCAPE benchmark (> 80,000 peptides
 from 27 repositories). This may overestimate discriminative power.
 
@@ -716,7 +716,7 @@ than the 10 originally planned. The path to 50%+ requires wet-lab data integrati
 ## Confidence Calibration
 
 This assessment is based on:
-- Internal benchmark AUROC = 0.8086 (n=88, bootstrap n=2000; phase3 synthesis gate = 0.7890; PR #65 Trp-weighted bonus update)  
+- Internal benchmark AUROC = 0.8047 (n=87, bootstrap n=2000; phase3 synthesis gate = 0.7846; PR #66 dedup fix)  
 - Literature hit rates for physchem AMP prediction (Loose et al. 2006; Tossi et al. 2002)  
 - Published serum stability data for short cationic peptides (Hilpert et al. 2006)  
 - D-amino acid t½ extension data (Wade et al. 1990, PNAS)  

diff --git a/docs/METHODS.md b/docs/METHODS.md
@@ -316,35 +316,37 @@ Phase 2 benchmarks verified that the pipeline:
 - Produces stable rankings under repeated runs (reproducibility)
 - Shows performance degradation when key scoring dimensions are ablated
 
-**Retrospective AUROC — pipeline.yaml (v0.7.x):** `0.8086` (95% CI: 0.7149–0.8957, n=2000
-bootstrap). Positive-vs-negative separation on the 44-AMP + 44-background benchmark set using
+**Retrospective AUROC — pipeline.yaml (v0.7.x):** `0.8047` (95% CI: 0.7072–0.8922, n=2000
+bootstrap). Positive-vs-negative separation on the 43-AMP + 44-background benchmark set using
 the full ensemble scorer with pipeline.yaml weights. Historical progression: 0.7926 → 0.8138 →
-0.8164 (PRs #48–54) → 0.8086 (PR #65: Trp-weighted aromatic bonus, safety abs() fix). The minor
-decrease from 0.8164 reflects that some random-background decoys contain Trp residues and benefit
-from the Trp bonus; this is within bootstrap CI width and the bonus is scientifically correct.
+0.8164 (PRs #48–54) → 0.8086 (PR #65: Trp-weighted aromatic bonus, safety abs() fix) →
+0.8047 (PR #66: removed duplicate GIGKFLHSAKKFGKAFVGEIMNS / REF-GIG-001 from validation set;
+43 unique AMPs used). The minor decrease from 0.8086 reflects that the removed entry (magainin-2)
+is a high-ranking AMP; fewer true positives at the top slightly reduce AUROC. Scientifically
+more accurate than the pre-dedup estimate.
 
-**Retrospective AUROC — phase3.yaml (synthesis gate, v0.7.x):** `0.7890` (95% CI: 0.6932–0.8784,
+**Retrospective AUROC — phase3.yaml (synthesis gate, v0.7.x):** `0.7846` (95% CI: 0.6829–0.8747,
 n=2000 bootstrap). Phase3 uses re-weighted ensemble scores (activity=0.35, safety=0.30,
 synthesis=0.20, novelty=0.15 vs pipeline.yaml activity=0.40, safety=0.25, synthesis=0.15,
 novelty=0.20) and a stricter safety gate (max_safety_risk=0.40). The AUROC is lower than
 pipeline.yaml because the higher safety weight down-ranks some literature AMPs that have
 hemolysis risk, which is scientifically correct behaviour for a synthesis gate. Interpretation:
-**STRONG** (AUROC > 0.70). AUPRC = 0.8301 (+0.3301 above random baseline of 0.50).
-Recall@10 = 0.23, Recall@20 = 0.43, Recall@44 = 0.70.
+**STRONG** (AUROC > 0.70). AUPRC = 0.8240 (+0.3297 above random baseline of 0.4943).
+Recall@10 = 0.23, Recall@20 = 0.44, Recall@43 = 0.70.
 
-> **Important:** The synthesis selection gate uses phase3.yaml. The phase3 AUROC (0.7890) is the
-> operationally relevant benchmark. pipeline.yaml AUROC (0.8086) is the full-ensemble reference.
-> Both point estimates exceed the AUROC > 0.70 synthesis gate. Note that with n=88 sequences the
+> **Important:** The synthesis selection gate uses phase3.yaml. The phase3 AUROC (0.7846) is the
+> operationally relevant benchmark. pipeline.yaml AUROC (0.8047) is the full-ensemble reference.
+> Both point estimates exceed the AUROC > 0.70 synthesis gate. Note that with n=87 sequences the
 > 95% CI spans the gate boundary; synthesis decisions are made on point estimates, which is standard
 > practice at this sample size — the CI reflects sampling uncertainty, not model unreliability.
 
-**AUPRC (v0.7.x):** `0.8495` for pipeline.yaml (+0.3495 above the random baseline of 0.50).
+**AUPRC (v0.7.x):** `0.8443` for pipeline.yaml (+0.2943 above the random baseline of 0.4943).
 Area Under Precision-Recall Curve is computed alongside AUROC and reported by `make validate-scoring`.
 AUPRC is preferable to AUROC for class-imbalanced datasets because it emphasises precision
 at the operating point actually used for candidate selection. Random baseline = dataset
-prevalence (0.50 for the balanced demo set). Implemented with pessimistic tie-breaking
-(negatives sort first on tied scores) to match sklearn `average_precision_score` convention
-and avoid inflation to 1.0 for constant-score classifiers.
+prevalence (43/87 ≈ 0.4943 for the slightly unbalanced post-dedup set). Implemented with
+pessimistic tie-breaking (negatives sort first on tied scores) to match sklearn
+`average_precision_score` convention and avoid inflation to 1.0 for constant-score classifiers.
 
 **To reproduce:** `make validate-scoring` (pipeline.yaml weights) or
 `make validate-scoring-phase3` (synthesis gate weights, phase3.yaml) or
@@ -362,8 +364,9 @@ and avoid inflation to 1.0 for constant-score classifiers.
 | #53 | Safety uses charge_density_ph74; stronger cytotox_penalty in pilot | pilot ranking |
 | #58 | AUPRC added; SEED-009 (Bac2A) + SEED-010 (KR-12) added | +2 scaffolds |
 | #65 | Trp-weighted aromatic bonus (1.5× vs Phe/Tyr); safety abs() fix | 0.8164→0.8086 |
+| #66 | Remove duplicate REF-GIG-001 (magainin-2 counted twice in validation set) | 0.8086→0.8047 |
 
-**Limitation:** Benchmarks use a small, curated demo dataset (44 AMPs + 44 background). They
+**Limitation:** Benchmarks use a small, curated demo dataset (43 AMPs + 44 background). They
 do not validate against large independent AMP databases. Real retrospective validation against
 APD3-scale data (> 3,000 AMPs, cluster-split) is required before strong claims of predictive
 power.

diff --git a/examples/validation/known_amps.csv b/examples/validation/known_amps.csv
@@ -24,7 +24,6 @@ REF-CEC-001,KWKLFKKIEKVGQNIRDGIIKAGPAV,cecropin_a_fragment,Steiner_1981_PNAS,1
 REF-CEC-002,KWKIFKKIEKVGRNVRDGIIKAGP,cecropin_b_fragment,Hultmark_1980_Eur_J_Biochem,1
 REF-MEL-001,GIGAVLKVLTTGLPALISWIKRKRQQ,melittin,Habermann_1972,1
 REF-KWK-001,KWKLFKKIGAVLKVL,template_seed_1,Oren_1997_Biochemistry,1
-REF-GIG-001,GIGKFLHSAKKFGKAFVGEIMNS,template_seed_2,Zasloff_1987_PNAS,1
 REF-MAG-004,KKLKKFGLRIINKIVAAL,magainin_helix,Wieprecht_1997,1
 REF-BMA-001,GRFKRFRKKFKKLFKKLSP,bmap_fragment,Skerlavaj_1999,1
 REF-LLL-001,LLRWPWWPWRRK,omiganan_analog,Sader_2004,1

diff --git a/examples/validation/scrambled_decoys.csv b/examples/validation/scrambled_decoys.csv
@@ -24,7 +24,6 @@ DECOY-CEC-001,KRWNVKKIIQKGAPDIKIGEFLVKGA,shuffled_cecropin_a_fragment,REF-CEC-00
 DECOY-CEC-002,KAGIKDIRIRIKGGWPNVKEKFVK,shuffled_cecropin_b_fragment,REF-CEC-002,0
 DECOY-MEL-001,ILTLIKTLIGQALGWKVPSQARVRKG,shuffled_melittin,REF-MEL-001,0
 DECOY-KWK-001,FALKGWKKLVLIKVK,shuffled_template_seed_1,REF-KWK-001,0
-DECOY-GIG-001,KKGHASFIFIGLEKNSVFMGAKG,shuffled_template_seed_2,REF-GIG-001,0
 DECOY-MAG-004,ALLKLAFKKKVGKRIIIN,shuffled_magainin_helix,REF-MAG-004,0
 DECOY-BMA-001,KKGRKPLFFRLFKRKKSKF,shuffled_bmap_fragment,REF-BMA-001,0
 DECOY-LLL-001,WLWPRLWKPRWR,shuffled_omiganan_analog,REF-LLL-001,0

diff --git a/src/openamp_foundry/benchmark/retrospective.py b/src/openamp_foundry/benchmark/retrospective.py
@@ -3,7 +3,7 @@
 Two complementary benchmark modes:
 
   STANDARD (primary gate):
-    Positives — 44 confirmed literature AMPs
+    Positives — 43 confirmed literature AMPs (unique; REF-GIG-001 duplicate removed in PR #66)
     Negatives — 44 length-matched peptides drawn from UniProt Swiss-Prot
                 background amino acid frequencies (RNG seed=43)
     Tests: can the model distinguish AMP-like composition/structure from
@@ -12,8 +12,8 @@
     Gate: AUROC > 0.70 → proceed; 0.55–0.70 → caution; < 0.55 → STOP.
 
   STRICT (order-sensitivity test, secondary):
-    Positives — 44 confirmed literature AMPs
-    Negatives — 44 per-sequence composition-matched shuffled decoys (RNG seed=42)
+    Positives — 43 confirmed literature AMPs
+    Negatives — 43 per-sequence composition-matched shuffled decoys (RNG seed=42)
     Tests: does the model have order-dependent signal beyond composition?
     Expected AUROC: 0.50–0.65 for any composition-heavy scorer. This is
     intentionally difficult — it tests ONLY the hydrophobic moment term
@@ -204,7 +204,7 @@ def run_retrospective_benchmark(
     labels_ranked = [r["label"] for r in rows]
 
     if recall_ks is None:
-        recall_ks = [10, 20, 44]
+        recall_ks = [10, 20, 43]
     recall = {f"recall_at_{k}": round(_recall_at_k(labels_ranked, k), 4) for k in recall_ks}
 
     random_auroc = 0.5

diff --git a/src/openamp_foundry/cli.py b/src/openamp_foundry/cli.py
@@ -542,7 +542,7 @@ def _run_validate_scoring(args: argparse.Namespace) -> int:
         "auroc_above_random": result["auroc_above_random"],
         "recall_at_10": result.get("recall_at_10"),
         "recall_at_20": result.get("recall_at_20"),
-        "recall_at_44": result.get("recall_at_44"),
+        "recall_at_43": result.get("recall_at_43"),
         "interpretation": result["interpretation"],
         "out": args.out,
     }
@@ -988,7 +988,7 @@ def _score(seq: str) -> dict:
         "",
         "This calibration uses the same scoring model as candidate nomination. It is not",
         "independent validation — it confirms internal consistency, not external predictive power.",
-        "The AUROC benchmark (AUROC=0.8037 on 44 literature AMPs vs 44 background peptides) is",
+        "The AUROC benchmark (AUROC=0.8047 on 43 literature AMPs vs 44 background peptides) is",
         "the appropriate independent validation.",
     ]
 

diff --git a/tests/test_benchmark_evaluate.py b/tests/test_benchmark_evaluate.py
@@ -69,9 +69,9 @@ def test_expected_random_recall_k_less_than_positives(self):
         assert abs(result - 0.1) < 0.001, f"Expected 0.1, got {result}"
 
     def test_random_recall_real_benchmark_scale(self):
-        # 44 AMPs, 88 total candidates, k=8 → E[recall@8] = 8/88 ≈ 0.0909
-        result = random_recall_at_k(n_candidates=88, n_positives=44, k=8)
-        assert abs(result - 8 / 88) < 0.001, f"Expected {8/88:.4f}, got {result}"
+        # 43 AMPs + 44 background = 87 total; k=8 → E[recall@8] = 8/87 ≈ 0.0920
+        result = random_recall_at_k(n_candidates=87, n_positives=43, k=8)
+        assert abs(result - 8 / 87) < 0.001, f"Expected {8/87:.4f}, got {result}"
 
     def test_zero_candidates_returns_zero(self):
         assert random_recall_at_k(0, 2, 2) == 0.0