{
  "date": "2026-10-13",
  "m4": {
    "protocol": {
      "lockedAtUTC": "2026-10-13T15:12:02.208334+00:00",
      "sourceSha256": "08527f163c57d28fc248a78eb7f9c262dafed686300727a4257df2e90d2ff68b",
      "source": "docs/validation/2026-10-13-response-reconciliation-v4/m4-aligned.parquet",
      "primaryMetric": "MAE, lower is better; selected before v5 evaluation to address poor absolute response prediction. Historical v4 selected Spearman.",
      "candidate": "Equal-weight ridge(alpha=100) plus gradient-boosted tree ensemble, 52 existing geometry features. This is an engineering candidate, not a novelty claim.",
      "selection": "12 configurations per family; inner gene-disjoint 80/20 split using unchanged aligned-v3 hash ordering; select lowest validation MAE. Refit training study; evaluate all genes of other study. Final fit selects mean inner MAE across folds, then fits both studies.",
      "features": [
        "nexon__mean",
        "nexon__max",
        "nexon__std",
        "tx_len__mean",
        "tx_len__max",
        "tx_len__std",
        "cds_len__mean",
        "cds_len__max",
        "cds_len__std",
        "utr5_len__mean",
        "utr5_len__max",
        "utr5_len__std",
        "utr3_len__mean",
        "utr3_len__max",
        "utr3_len__std",
        "stop_to_lastEJ__mean",
        "stop_to_lastEJ__max",
        "stop_to_lastEJ__std",
        "downstream_junctions__mean",
        "downstream_junctions__max",
        "downstream_junctions__std",
        "stop_upstream_50nt__mean",
        "stop_upstream_50nt__max",
        "stop_upstream_50nt__std",
        "max_exon_len__mean",
        "max_exon_len__max",
        "max_exon_len__std",
        "mean_exon_len__mean",
        "mean_exon_len__max",
        "mean_exon_len__std",
        "last_exon_len__mean",
        "last_exon_len__max",
        "last_exon_len__std",
        "cds_fraction__mean",
        "cds_fraction__max",
        "cds_fraction__std",
        "GC_tx__mean",
        "GC_tx__max",
        "GC_tx__std",
        "GC_utr5__mean",
        "GC_utr5__max",
        "GC_utr5__std",
        "GC_cds__mean",
        "GC_cds__max",
        "GC_cds__std",
        "GC_utr3__mean",
        "GC_utr3__max",
        "GC_utr3__std",
        "utr3_mfe_nt__mean",
        "utr3_mfe_nt__max",
        "utr3_mfe_nt__std",
        "isoform_count"
      ],
      "configurations": {
        "M4_v5": [
          {
            "max_depth": 2,
            "min_child_weight": 20,
            "reg_lambda": 10
          },
          {
            "max_depth": 2,
            "min_child_weight": 20,
            "reg_lambda": 100
          },
          {
            "max_depth": 2,
            "min_child_weight": 80,
            "reg_lambda": 10
          },
          {
            "max_depth": 2,
            "min_child_weight": 80,
            "reg_lambda": 100
          },
          {
            "max_depth": 3,
            "min_child_weight": 20,
            "reg_lambda": 10
          },
          {
            "max_depth": 3,
            "min_child_weight": 20,
            "reg_lambda": 100
          },
          {
            "max_depth": 3,
            "min_child_weight": 80,
            "reg_lambda": 10
          },
          {
            "max_depth": 3,
            "min_child_weight": 80,
            "reg_lambda": 100
          },
          {
            "max_depth": 5,
            "min_child_weight": 20,
            "reg_lambda": 10
          },
          {
            "max_depth": 5,
            "min_child_weight": 20,
            "reg_lambda": 100
          },
          {
            "max_depth": 5,
            "min_child_weight": 80,
            "reg_lambda": 10
          },
          {
            "max_depth": 5,
            "min_child_weight": 80,
            "reg_lambda": 100
          }
        ],
        "ExtraTrees": [
          {
            "min_samples_leaf": 5,
            "max_features": 0.5,
            "max_depth": 18
          },
          {
            "min_samples_leaf": 5,
            "max_features": 0.5,
            "max_depth": null
          },
          {
            "min_samples_leaf": 5,
            "max_features": 1.0,
            "max_depth": 18
          },
          {
            "min_samples_leaf": 5,
            "max_features": 1.0,
            "max_depth": null
          },
          {
            "min_samples_leaf": 10,
            "max_features": 0.5,
            "max_depth": 18
          },
          {
            "min_samples_leaf": 10,
            "max_features": 0.5,
            "max_depth": null
          },
          {
            "min_samples_leaf": 10,
            "max_features": 1.0,
            "max_depth": 18
          },
          {
            "min_samples_leaf": 10,
            "max_features": 1.0,
            "max_depth": null
          },
          {
            "min_samples_leaf": 20,
            "max_features": 0.5,
            "max_depth": 18
          },
          {
            "min_samples_leaf": 20,
            "max_features": 0.5,
            "max_depth": null
          },
          {
            "min_samples_leaf": 20,
            "max_features": 1.0,
            "max_depth": 18
          },
          {
            "min_samples_leaf": 20,
            "max_features": 1.0,
            "max_depth": null
          }
        ],
        "ridge": [
          {
            "alpha": 0.01
          },
          {
            "alpha": 0.1
          },
          {
            "alpha": 1.0
          },
          {
            "alpha": 10.0
          },
          {
            "alpha": 100.0
          },
          {
            "alpha": 1000.0
          },
          {
            "alpha": 10000.0
          },
          {
            "alpha": 100000.0
          },
          {
            "alpha": 1000000.0
          },
          {
            "alpha": 10000000.0
          },
          {
            "alpha": 100000000.0
          },
          {
            "alpha": 1000000000.0
          }
        ]
      },
      "freshHoldout": false,
      "superiorityEstablished": false,
      "limitations": [
        "Two repeatedly examined studies; development diagnostics only.",
        "Study and gene overlap differ: 7400 genes shared between studies.",
        "SLAM and BRIC assays, time/perturbation and feature-ascertainment differences persist.",
        "No test-based calibration or outcome filtering.",
        "Fixed 50/50 ridge/boost blend is explicitly compared with standalone ridge and tuned ExtraTrees."
      ]
    },
    "folds": [
      {
        "heldStudy": "NMDRHT",
        "genes": 10990,
        "overlappingGenes": 7400,
        "metrics": {
          "M4_v5": {
            "spearman": 0.45411379230757803,
            "mae": 0.5639923644861607,
            "r2": -0.09000941027995091
          },
          "ExtraTrees": {
            "spearman": 0.4594547414317151,
            "mae": 0.56460278036736,
            "r2": -0.08965009874549601
          },
          "ridge": {
            "spearman": 0.4474211485032731,
            "mae": 0.5655987582260991,
            "r2": -0.10327427061122418
          },
          "training_median": {
            "spearman": null,
            "mae": 0.6192952740562929,
            "r2": -0.32025102005434203
          }
        },
        "comparisons": [
          {
            "baseline": "ExtraTrees",
            "baselineMinusM4MAE": 0.0006104158811992555,
            "adjustedPairedCi": [
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              0.0033089874418685172
            ]
          },
          {
            "baseline": "ridge",
            "baselineMinusM4MAE": 0.0016063937399383166,
            "adjustedPairedCi": [
              -0.0008932818962835375,
              0.0042717170108161244
            ]
          }
        ],
        "selections": {
          "M4_v5": {
            "configurationId": 8,
            "innerMAE": 0.538660514189818
          },
          "ExtraTrees": {
            "configurationId": 4,
            "innerMAE": 0.5369374911481749
          },
          "ridge": {
            "configurationId": 4,
            "innerMAE": 0.5511719760842511
          }
        }
      },
      {
        "heldStudy": "Tani2012",
        "genes": 7687,
        "overlappingGenes": 7400,
        "metrics": {
          "M4_v5": {
            "spearman": 0.4305228439272508,
            "mae": 0.6790880283361039,
            "r2": -0.07707156843482554
          },
          "ExtraTrees": {
            "spearman": 0.4797730754387891,
            "mae": 0.652379919795169,
            "r2": -0.02761578497146111
          },
          "ridge": {
            "spearman": 0.40326883172921957,
            "mae": 0.6912682315411992,
            "r2": -0.10234315959144658
          },
          "training_median": {
            "spearman": null,
            "mae": 0.6711025223525823,
            "r2": -0.11712206651913015
          }
        },
        "comparisons": [
          {
            "baseline": "ExtraTrees",
            "baselineMinusM4MAE": -0.02670810854093497,
            "adjustedPairedCi": [
              -0.03302710745621495,
              -0.020412836651627676
            ]
          },
          {
            "baseline": "ridge",
            "baselineMinusM4MAE": 0.012180203205095275,
            "adjustedPairedCi": [
              0.009668255932807552,
              0.014705102187894626
            ]
          }
        ],
        "selections": {
          "M4_v5": {
            "configurationId": 9,
            "innerMAE": 0.47340129226801764
          },
          "ExtraTrees": {
            "configurationId": 2,
            "innerMAE": 0.4719610436304663
          },
          "ridge": {
            "configurationId": 4,
            "innerMAE": 0.48588149176879863
          }
        }
      }
    ],
    "finalModels": {
      "M4_v5": {
        "configurationId": 9,
        "sha256": "b565e5c5e1166764d44467442364e3f5011520f8f0438ae71eecb18142494266"
      },
      "ExtraTrees": {
        "configurationId": 4,
        "sha256": "9b3b0d9ca94e41580b6927dfb03efaa3221ac5ba65c154f5e3ea3a3867a82acf"
      },
      "ridge": {
        "configurationId": 4,
        "sha256": "1832ca7c0077b5c31291a7f5b7b36b428363ef6f466077dad169308a203dd9d4"
      }
    },
    "freshHoldout": false,
    "superiorityEstablished": false
  },
  "m5": {
    "Aliouat2019": {
      "genes": 5268,
      "oldNewTargetSpearman": 0.9938799440056189,
      "frozenV6SensitivitySpearman": {
        "M5_v6": -0.22098742378687888,
        "ExtraTrees": -0.21552350486861,
        "ridge": -0.17928825989808683
      }
    },
    "NMDRHT": {
      "genes": 4886,
      "oldNewTargetSpearman": 0.9947787958487141,
      "frozenV6SensitivitySpearman": {
        "M5_v6": -0.31316133309527877,
        "ExtraTrees": -0.3187425079076596,
        "ridge": -0.27510481399406317
      }
    },
    "crossStudy": {
      "genes": 4628,
      "correlations": {
        "RNA_log2FC": 0.22888937422415037,
        "RPF_log2FC": 0.3161451433586253,
        "TE": -0.18589706459586083,
        "target": -0.18859932464146273
      }
    }
  },
  "metadata": {
    "qualified": false,
    "outcomesOpened": false,
    "source": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE276918",
    "findings": [
      "Current GEO series and all six conflicting UPF1 sample records still have the 4h/8h title-versus-filename mismatch; records list May 31 2025 as last update.",
      "ENA experiment records SRX26053390/SRX26053393 and BioSample SAMN43580456 repeat GEO titles rather than provide an independent corrected sample map.",
      "Actinomycin-D is supported by the BioSample treatment and ENA library protocol, but the GEO summary still says alpha-amanitin. This strengthens one interpretation without resolving the provenance conflict.",
      "ENA submitted-file reports expose run IDs but no original submitted filenames or checksums for the two checked experiments. They do not resolve the time mapping.",
      "No publication identifier is linked in the current GEO series. A related author/title preprint was located through Europe PMC, but full text retrieval returned 403; its methods/supplement were not verified.",
      "Exact depletion duration, paired control timing and whether spike-ins were used for sequencing normalization remain unconfirmed."
    ],
    "conflicts": [
      {
        "title": "HCT116-UPF1,4h-1",
        "fileDescription": "I8-1_UPF1_1.fq.gz",
        "accession": "GSM8508966"
      },
      {
        "title": "HCT116-UPF1,4h-2",
        "fileDescription": "I8-2_UPF1_1.fq.gz",
        "accession": "GSM8508967"
      },
      {
        "title": "HCT116-UPF1,4h-3",
        "fileDescription": "I8-3_UPF1_1.fq.gz",
        "accession": "GSM8508968"
      },
      {
        "title": "HCT116-UPF1,8h-1",
        "fileDescription": "I4-1_UPF1_1.fq.gz",
        "accession": "GSM8508969"
      },
      {
        "title": "HCT116-UPF1,8h-2",
        "fileDescription": "I4-2_UPF1_1.fq.gz",
        "accession": "GSM8508970"
      },
      {
        "title": "HCT116-UPF1,8h-3",
        "fileDescription": "I4-3_UPF1_1.fq.gz",
        "accession": "GSM8508971"
      }
    ],
    "decision": "Do not fit decay rates or benchmark GSE276918 until an authoritative sample map resolves these issues. New outcome counts remain unopened."
  },
  "search": {
    "qualifiedStudies": 0,
    "freshTestRun": false,
    "scope": "Targeted human UPF1 RNA/RPF searches and primary full-text lineage review; not exhaustive. Rechecked recent papers and the eRF1/UPF1 study trail.",
    "sources": [
      {
        "name": "Feedback from the Nascent Chain (2026)",
        "url": "https://pmc.ncbi.nlm.nih.gov/articles/PMC12776465/",
        "decision": "UPF1 abundance analysis cites Boehm/NMDRHT in reference 37; no new matched human UPF1 RNA/RPF study established."
      },
      {
        "name": "Elongationless start-stop elements (2026)",
        "url": "https://pmc.ncbi.nlm.nih.gov/articles/PMC13338717/",
        "decision": "Methods use published UPF1 datasets; reference 73 is Boehm/NMDRHT. A new publication is not a fresh experiment."
      },
      {
        "name": "Hidden stop codons / GSE303542",
        "url": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE303542",
        "decision": "Previously reviewed human RPF arm is eRF1 depletion; it does not supply matched human UPF1 RNA/RPF."
      }
    ]
  },
  "superiorityEstablished": false,
  "m5Conclusion": "Separate RNA/RPF DESeq2 fits do not resolve negative UPF1 transfer. Approximate target ranks remain nearly identical to the joint estimator. This sensitivity analysis cannot identify a causal mechanism or repair missing time-matched controls. Frozen v6 predictions were rescored without retraining or changing targets in the serving model.",
  "m5Method": "Same original source count matrices, assay-specific median-ratio normalization, and common count-filter rules. Fit depleted-versus-control separately within RNA and RPF; TE = RPF log2FC minus RNA log2FC. No significance filter; evaluation membership is the unchanged v6 cohort. This omits cross-assay pairing, so it is a sensitivity diagnostic, not a replacement for the paired Aliouat estimator.",
  "nextStep": "Obtain corrected GSE276918 labels and sample-level timing/normalization metadata; obtain a genuinely independent human UPF1 RNA/RPF cohort. Harmonized raw-read re-quantification and author confirmation remain necessary before attributing the transfer failure to assay differences."
}