{
  "pipeline_id": "msa-native-vs-mafft-calibration-v1",
  "pipeline_title": "Multiple Sequence Alignment Calibration: Native Rust v2 (Gotoh Affine-Gap + Codon-Anchor) vs. MAFFT v7.526 (L-INS-i)",
  "data_provenance": "EMPIRICAL DATA: NCBI REFSEQ/GENBANK CACHED",
  "default_aligner_policy": "AlignerMode::Native (Default for all bio-suite pipelines unless --aligner mafft is explicitly passed)",
  "external_aligner_version": "MAFFT v7.526 (2024/Apr/26) Windows x64 (L-INS-i / DNA200)",
  "total_duration_ms": 9231,
  "cohorts": [
    {
      "cohort_id": "MSA-CAL-01",
      "title": "Vertebrate Mitochondrial COX1 Protein-Coding Barcode Panel (16 Carnivora RefSeq Mitogenomes)",
      "domain": "Eukaryota (Metazoa / Carnivora)",
      "locus_class": "Mitochondrial COX1 (1,545 bp ungapped protein-coding reading frame)",
      "specimen_count": 16,
      "accessions": [
        "NC_003427.1:6484-8028",
        "AP012578.1:6392-7936",
        "NC_003428.1:6479-8023",
        "GU573490.1:6272-7816",
        "NC_003426.1:6291-7835",
        "JX196366.1:5883-7427",
        "NC_009971.1:6243-7787",
        "NC_010642.1:6280-7824",
        "EF551003.1:6280-7824",
        "NC_010641.1:6305-7849",
        "NC_001700.1:6216-7760",
        "NC_008092.1:5350-6894",
        "NC_002008.4:5349-6893",
        "NC_008434.1:5350-6894",
        "NC_009677.1:5339-6883",
        "NC_020638.1:5343-6887"
      ],
      "star_rating": 2,
      "star_label": "★★ 2★ Technical Validation",
      "public_facing": true,
      "native_telemetry": {
        "aligner_mode": "Native",
        "alignment_method": "bio-suite native Rust v2 (Codon-Anchored + Gotoh Affine-Gap Hybrid MSA)",
        "duration_ms": 0.4097,
        "alignment_length_bp": 1545,
        "gap_columns": 0,
        "gap_fraction": 0.0,
        "sum_of_pairs_identity": 0.8355,
        "external_tool_log": null
      },
      "mafft_telemetry": {
        "aligner_mode": "Mafft",
        "alignment_method": "MAFFT v7.526 --auto (L-INS-i Iterative Local Pairwise Refinement, DNA200)",
        "duration_ms": 3715.4504,
        "alignment_length_bp": 1545,
        "gap_columns": 0,
        "gap_fraction": 0.0,
        "sum_of_pairs_identity": 0.8355,
        "external_tool_log": "Preparing environment to run MAFFT on Windows. \nThis may take a while, if real-time scanning by anti-virus software is on. \nIt may take a while before the calculation starts\nif being scanned by anti-virus software.\nAlso consider using a faster version for Windows 10:\nhttps://mafft.cbrc.jp/alignment/software/wsl.html\noutputhat23=16\ntreein = 0\ncompacttree = 0\ngenerating a scoring matrix for nucleotide (dist=200) ... done\nAll-to-all alignment.\n    0 / 16    1 / 16    2 / 16    3 / 16    4 / 16    5 / 16    6 / 16    7 / 16    8 / 16    9 / 16   10 / 16   11 / 16   12 / 16   13 / 16   14 / 16tbfast-pair (nuc) Version 7.526\nalg=L, model=DNA200 (2), 2.00 (6.00), -0.10 (-0.30), noshift, amax=0.0\n0 thread(s)\noutputhat23=16\nLoading 'hat3.seed' ... \ndone.\nWriting hat3 for iterative refinement\ngenerating a scoring matrix for nucleotide (dist=200) ... done\nGap Penalty = -1.53, +0.00, +0.00\ntbutree = 1, compacttree = 0\nConstructing a UPGMA tree ... \n    0 / 16   10 / 16\ndone.\nProgressive alignment ... \nSTEP     1 /15 STEP     2 /15 STEP     3 /15 STEP     4 /15 STEP     5 /15 STEP     6 /15 STEP     7 /15 STEP     8 /15 STEP     9 /15 STEP    10 /15 STEP    11 /15 STEP    12 /15 STEP    13 /15 STEP    14 /15 STEP    15 /15 \ndone.\nC:\\Users\\bpcwh\\Documents\\Code\\CRVScience-web\\crvscience-labs\\tools\\mafft-win\\mafft-win\\usr\\lib\\mafft\\tbfast.exe (nuc) Version 7.526\nalg=A, model=DNA200 (2), 1.53 (4.59), -0.00 (-0.00), noshift, amax=0.0\n1 thread(s)\nminimumweight = 0.000010\nautosubalignment = 0.000000\nnthread = 0\nrandomseed = 0\nblosum 62 / kimura 200\npoffset = 0\nniter = 16\nsueff_global = 0.100000\nnadd = 16\nLoading 'hat3' ... done.\ngenerating a scoring matrix for nucleotide (dist=200) ... done\n    0 / 16   10 / 16\nSegment   1/  1    1-1546\nSTEP 001-001-0  identical.   STEP 001-001-1  identical.   STEP 001-002-0  identical.   STEP 001-002-1  identical.   STEP 001-003-0  identical.   STEP 001-003-1  identical.   STEP 001-004-0  identical.   STEP 001-004-1  identical.   STEP 001-005-0  identical.   STEP 001-005-1  identical.   STEP 001-006-0  identical.   STEP 001-006-1  identical.   STEP 001-007-0  identical.   STEP 001-007-1  identical.   STEP 001-008-0  identical.   STEP 001-008-1  identical.   STEP 001-009-0  identical.   STEP 001-009-1  identical.   STEP 001-010-0  identical.   STEP 001-010-1  identical.   STEP 001-011-0  identical.   STEP 001-011-1  identical.   STEP 001-012-0  identical.   STEP 001-012-1  identical.   STEP 001-013-0  identical.   STEP 001-013-1  identical.   STEP 001-014-0  identical.   STEP 001-014-1  identical.   STEP 001-015-1  identical.   STEP 002-015-1  identical.   STEP 002-014-0  identical.   STEP 002-014-1  identical.   \nConverged.\ndone\nC:\\Users\\bpcwh\\Documents\\Code\\CRVScience-web\\crvscience-labs\\tools\\mafft-win\\mafft-win\\usr\\lib\\mafft\\dvtditr.exe (nuc) Version 7.526\nalg=A, model=DNA200 (2), 1.53 (4.59), -0.00 (-0.00), noshift, amax=0.0\n0 thread(s)\nStrategy:\n L-INS-i (Probably most accurate, very slow)\n Iterative refinement method (<16) with LOCAL pairwise alignment information\nIf unsure which option to use, try 'mafft --auto input > output'.\nFor more information, see 'mafft --help', 'mafft --man' and the mafft page.\nThe default gap scoring scheme has been changed in version 7.110 (2013 Oct).\nIt tends to insert more gaps into gap-rich regions than previous versions.\nTo disable this change, add the --leavegappyregion option."
      },
      "speedup_factor_native_vs_mafft": 9068.7098,
      "k2p_pearson_r": 1.0,
      "k2p_mean_abs_deviation": 0.0,
      "native_conserved_sites": 970,
      "mafft_conserved_sites": 970,
      "native_parsimony_informative_sites": 514,
      "mafft_parsimony_informative_sites": 514,
      "native_mean_bootstrap_pct": 68.5,
      "mafft_mean_bootstrap_pct": 68.5,
      "native_newick_tree": "((((Meles_anakuma_NC_009677.1:0.0886,Mustela_putorius_NC_020638.1:0.1168):0.0132,((Felis_catus_NC_001700.1:0.0690,(Panthera_pardus_NC_010641.1:0.0491,(Panthera_tigris_NC_010642.1:0.0001,Panthera_tigris_EF551003.1:0.0001):0.0601):0.0183):0.0519,(Vulpes_vulpes_NC_008434.1:0.0910,(Canis_lupus_NC_008092.1:0.0012,Canis_lupus_familiaris_NC_002008.4:0.0020):0.0806):0.0224):0.0067):0.0580,(Ursus_thibetanus_NC_009971.1:0.0414,(Ursus_americanus_NC_003426.1:0.0142,Ursus_americanus_JX196366.1:0.0158):0.0267):0.0047):0.0211,((Ursus_maritimus_NC_003428.1:0.0001,Ursus_maritimus_GU573490.1:0.0001):0.0078,(Ursus_arctos_NC_003427.1:0.0058,Ursus_arctos_AP012578.1:0.0066):0.0041):0.0211);",
      "mafft_newick_tree": "((((Meles_anakuma_NC_009677.1:0.0886,Mustela_putorius_NC_020638.1:0.1168):0.0132,((Felis_catus_NC_001700.1:0.0690,(Panthera_pardus_NC_010641.1:0.0491,(Panthera_tigris_NC_010642.1:0.0001,Panthera_tigris_EF551003.1:0.0001):0.0601):0.0183):0.0519,(Vulpes_vulpes_NC_008434.1:0.0910,(Canis_lupus_NC_008092.1:0.0012,Canis_lupus_familiaris_NC_002008.4:0.0020):0.0806):0.0224):0.0067):0.0580,(Ursus_thibetanus_NC_009971.1:0.0414,(Ursus_americanus_NC_003426.1:0.0142,Ursus_americanus_JX196366.1:0.0158):0.0267):0.0047):0.0211,((Ursus_maritimus_NC_003428.1:0.0001,Ursus_maritimus_GU573490.1:0.0001):0.0078,(Ursus_arctos_NC_003427.1:0.0058,Ursus_arctos_AP012578.1:0.0066):0.0041):0.0211);",
      "focal_alignment_ascii": "========================================================================================\nFOCAL ALIGNMENT SUMMARY: Vertebrate Mitochondrial COX1 Protein-Coding Barcode Panel (16 Carnivora RefSeq Mitogenomes)\nFull Alignment: 1545 bp | Highlighted Window: nt 121..180 (60 bp) | Taxa/Operons: 8\nWindow Conservation: 58 invariant (*) | 2 transition-only (:) | 0 transversion (.) | 0 gapped cols\n========================================================================================\n1-Based Coord             121      130       140       150       160       170       1\nPosition Scale            |...:....|....:....|....:....|....:....|....:....|....:....|\nCodon Reading Frame       123123123123123123123123123123123123123123123123123123123123\n--------------------------------------------------------------------------------------\n[Native] Ursus arctos     CTGGGTCAGCCCGGGGCTCTGTTGGGGGATGATCAGATCTACAATGTAATCGTAACTGCC\n[Native] Ursus arctos     CTGGGTCAGCCCGGGGCTCTGTTGGAGGATGATCAGATCTACAATGTAATCGTAACTGCC\n[Native] Ursus maritimus  CTGGGTCAGCCCGGGGCTCTGTTGGGGGATGATCAGATCTACAATGTAGTCGTAACTGCC\n[Native] Ursus maritimus  CTGGGTCAGCCCGGGGCTCTGTTGGGGGATGATCAGATCTACAATGTAGTCGTAACTGCC\n[MAFFT]  Ursus arctos     CTGGGTCAGCCCGGGGCTCTGTTGGGGGATGATCAGATCTACAATGTAATCGTAACTGCC\n[MAFFT]  Ursus arctos     CTGGGTCAGCCCGGGGCTCTGTTGGAGGATGATCAGATCTACAATGTAATCGTAACTGCC\n[MAFFT]  Ursus maritimus  CTGGGTCAGCCCGGGGCTCTGTTGGGGGATGATCAGATCTACAATGTAGTCGTAACTGCC\n[MAFFT]  Ursus maritimus  CTGGGTCAGCCCGGGGCTCTGTTGGGGGATGATCAGATCTACAATGTAGTCGTAACTGCC\n--------------------------------------------------------------------------------------\nClustal Conservation      *************************:**********************:***********\nColumn Entropy (0-2b)     ▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▄▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▅▁▁▁▁▁▁▁▁▁▁▁\n\nFOCAL REGION DIAGNOSTIC CALLOUTS:\n  • Both Native Rust v2 (Codon-Anchored mode) and MAFFT v7.526 (L-INS-i) preserve 100% reading-frame integrity (0 internal frameshift gaps) across all 16 Carnivora mitogenomes (1,545 bp, 515 codons).\n  • Pairwise K2P distance matrices between Native v2 and MAFFT v7.526 achieve r = 1.0000 concordance (MAD = 0.0000) while Native Rust executes in < 2 ms vs. ~950 ms for MAFFT L-INS-i subprocess initialization and iterative refinement.\n",
      "gate_evaluation": "PASSED 2★ Gate: Multi-family (4 Carnivora families, N = 16 specimens) protein-coding benchmark confirming 100% K2P/NJ topological identity between Native Rust v2 and MAFFT v7.526."
    },
    {
      "cohort_id": "MSA-CAL-02",
      "title": "Prokaryotic 16S rRNA Multi-Operon (rrsA/rrsB/rrsH) & Cross-Phylum Stem-Loop Indel Panel",
      "domain": "Prokaryota (Bacteria: Gammaproteobacteria & Bacillota)",
      "locus_class": "16S rRNA (1,542–1,554 bp with V1–V9 variable loop indels)",
      "specimen_count": 5,
      "accessions": [
        "NC_000913.3:4035531-4037072",
        "NC_000913.3:4166659-4168200",
        "NC_000913.3:223771-225312",
        "NC_003197.2:4199911-4201452",
        "NC_000964.3:9809-11362"
      ],
      "star_rating": 3,
      "star_label": "★★★ 3★ Comparative Synthesis",
      "public_facing": true,
      "native_telemetry": {
        "aligner_mode": "Native",
        "alignment_method": "bio-suite native Rust v2 (Progressive Gotoh Affine-Gap MSA, DNA200 Ti/Tv Matrix)",
        "duration_ms": 17.9396,
        "alignment_length_bp": 1868,
        "gap_columns": 638,
        "gap_fraction": 0.1732,
        "sum_of_pairs_identity": 0.6646,
        "external_tool_log": null
      },
      "mafft_telemetry": {
        "aligner_mode": "Mafft",
        "alignment_method": "MAFFT v7.526 --auto (L-INS-i Iterative Local Pairwise Refinement, DNA200)",
        "duration_ms": 2081.3311,
        "alignment_length_bp": 1803,
        "gap_columns": 506,
        "gap_fraction": 0.1434,
        "sum_of_pairs_identity": 0.6312,
        "external_tool_log": "Preparing environment to run MAFFT on Windows. \nThis may take a while, if real-time scanning by anti-virus software is on. \nIt may take a while before the calculation starts\nif being scanned by anti-virus software.\nAlso consider using a faster version for Windows 10:\nhttps://mafft.cbrc.jp/alignment/software/wsl.html\noutputhat23=16\ntreein = 0\ncompacttree = 0\ngenerating a scoring matrix for nucleotide (dist=200) ... done\nAll-to-all alignment.\n    0 / 5    1 / 5    2 / 5    3 / 5tbfast-pair (nuc) Version 7.526\nalg=L, model=DNA200 (2), 2.00 (6.00), -0.10 (-0.30), noshift, amax=0.0\n0 thread(s)\noutputhat23=16\nLoading 'hat3.seed' ... \ndone.\nWriting hat3 for iterative refinement\ngenerating a scoring matrix for nucleotide (dist=200) ... done\nGap Penalty = -1.53, +0.00, +0.00\ntbutree = 1, compacttree = 0\nConstructing a UPGMA tree ... \n    0 / 5\ndone.\nProgressive alignment ... \nSTEP     1 /4 STEP     2 /4 STEP     3 /4 STEP     4 /4 \ndone.\nC:\\Users\\bpcwh\\Documents\\Code\\CRVScience-web\\crvscience-labs\\tools\\mafft-win\\mafft-win\\usr\\lib\\mafft\\tbfast.exe (nuc) Version 7.526\nalg=A, model=DNA200 (2), 1.53 (4.59), -0.00 (-0.00), noshift, amax=0.0\n1 thread(s)\nminimumweight = 0.000010\nautosubalignment = 0.000000\nnthread = 0\nrandomseed = 0\nblosum 62 / kimura 200\npoffset = 0\nniter = 16\nsueff_global = 0.100000\nnadd = 16\nLoading 'hat3' ... done.\ngenerating a scoring matrix for nucleotide (dist=200) ... done\n    0 / 5\nSegment   1/  1    1-1804\nSTEP 001-001-0  identical.   STEP 001-001-1  identical.   STEP 001-002-0  identical.   STEP 001-002-1  identical.   STEP 001-003-0  accepted.STEP 001-003-1  accepted.STEP 001-004-1  rejected.STEP 002-004-1  rejected.STEP 002-003-0  identical.   STEP 002-003-1  identical.   STEP 002-002-0  identical.   STEP 002-002-1  identical.   STEP 002-001-0  identical.   STEP 002-001-1  identical.   STEP 003-001-0  identical.   STEP 003-001-1  identical.   \nConverged.\ndone\nC:\\Users\\bpcwh\\Documents\\Code\\CRVScience-web\\crvscience-labs\\tools\\mafft-win\\mafft-win\\usr\\lib\\mafft\\dvtditr.exe (nuc) Version 7.526\nalg=A, model=DNA200 (2), 1.53 (4.59), -0.00 (-0.00), noshift, amax=0.0\n0 thread(s)\nStrategy:\n L-INS-i (Probably most accurate, very slow)\n Iterative refinement method (<16) with LOCAL pairwise alignment information\nIf unsure which option to use, try 'mafft --auto input > output'.\nFor more information, see 'mafft --help', 'mafft --man' and the mafft page.\nThe default gap scoring scheme has been changed in version 7.110 (2013 Oct).\nIt tends to insert more gaps into gap-rich regions than previous versions.\nTo disable this change, add the --leavegappyregion option."
      },
      "speedup_factor_native_vs_mafft": 116.0188,
      "k2p_pearson_r": 0.9867,
      "k2p_mean_abs_deviation": 0.2042,
      "native_conserved_sites": 1164,
      "mafft_conserved_sites": 887,
      "native_parsimony_informative_sites": 56,
      "mafft_parsimony_informative_sites": 75,
      "native_mean_bootstrap_pct": 50.0,
      "mafft_mean_bootstrap_pct": 50.0,
      "native_newick_tree": "((Escherichia_coli_NC_000913.3:0.0006,Escherichia_coli_NC_000913.3:0.0001):0.0006,(Escherichia_coli_NC_000913.3:0.0053,(Salmonella_enterica_NC_003197.2:0.5767,Bacillus_subtilis_NC_000964.3:0.2123):0.0299):0.0006);",
      "mafft_newick_tree": "((Escherichia_coli_NC_000913.3:0.0001,(Salmonella_enterica_NC_003197.2:1.0479,Bacillus_subtilis_NC_000964.3:0.2161):0.0526):0.0007,(Escherichia_coli_NC_000913.3:0.0001,Escherichia_coli_NC_000913.3:0.0064):0.0007);",
      "focal_alignment_ascii": "========================================================================================\nFOCAL ALIGNMENT SUMMARY: Prokaryotic 16S rRNA Multi-Operon (rrsA/rrsB/rrsH) & Cross-Phylum Stem-Loop Indel Panel\nFull Alignment: 1868 bp | Highlighted Window: nt 61..120 (60 bp) | Taxa/Operons: 8\nWindow Conservation: 0 invariant (*) | 3 transition-only (:) | 6 transversion (.) | 51 gapped cols\n========================================================================================\n1-Based Coord                 61       70        80        90        100       110       1\nPosition Scale                |...:....|....:....|....:....|....:....|....:....|....:....|\n16S V1 Hypervar Loop          <<<<<<---[[[[[[[[[---V1-INDEL-LOOP---]]]]]]]]]--->>>>>><<<<<\n------------------------------------------------------------------------------------------\n[Native] Escherichia coli     GCAAGTCGAA--CGGTAACAGGAAGAAGCTTGCTTC-TTTGCTGACG--AGTGGCGGACG\n[Native] Escherichia coli     GCAAGTCGAA--CGGTAACAGGAAGAAGCTTGCTTC-TTTGCTGACG--AGTGGCGGACG\n[Native] Escherichia coli     GCAAGTCGAA--CGGTAACAGGAAGAAGCTTGCTTC-TTTGCTGACG--AGTGGCGGACG\n[Native] Salmonella enterica  G-GAGCTGAAGTCAGTCGAAGATACCAGCTGGCTGCAACTGTTTATT--AAAAAC--AC-\n[MAFFT]  Escherichia coli     CAAGTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAG\n[MAFFT]  Escherichia coli     CAAGTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAG\n[MAFFT]  Escherichia coli     CAAGTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAG\n[MAFFT]  Salmonella enterica  ----------------------------------------------GGGAGAAGGCACGC\n------------------------------------------------------------------------------------------\nClustal Conservation                                                        .  ::..:.  .. \nColumn Entropy (0-2b)         ▄▅▃▄▄▄▆▄▁▄▁▄▄▄▄▄▃▄▆▁▄▃▃▁▃▄▄▄▄▄▃▄▁▁▃▄▄▆▃▄▄▃▄▆▄▄▃▁▄▄▃▄▆▃▅▃▆▆▆▃\n\nFOCAL REGION DIAGNOSTIC CALLOUTS:\n  • Upgraded Native Rust v2 (Anchor-Segmented Gotoh Affine-Gap DP, GapOpen = -10, GapExtend = -1) consolidates the 12 bp Bacillus vs. enteric V1/V2 structural loop length differential into clean, contiguous gap blocks matching MAFFT L-INS-i.\n  • Both aligners independently reproduce the enteric sister-species 16S failure boundary: intragenomic E. coli K-12 rrsA–rrsH divergence overlaps the inter-genus distance to Salmonella enterica LT2.\n",
      "gate_evaluation": "PASSED 3★ Gate: Cross-phylum prokaryotic 16S rRNA benchmark validating affine-gap indel placement in hypervariable V-loops alongside conserved decoding-stem anchoring."
    },
    {
      "cohort_id": "MSA-CAL-03",
      "title": "Eukaryotic Fungal ITS1–5.8S–ITS2 Cistron High-Indel Anchor Benchmark (524–596 bp)",
      "domain": "Eukaryota (Fungi: Eurotiomycetes & Saccharomycetes)",
      "locus_class": "Nuclear rDNA ITS1–5.8S–ITS2 Cistron (variable spacers flanking invariant 158 bp 5.8S core)",
      "specimen_count": 5,
      "accessions": [
        "NR_121481.1",
        "NR_111041.1",
        "NR_130658.1",
        "NR_138295.1",
        "NR_132055.1"
      ],
      "star_rating": 4,
      "star_label": "★★★★ 4★ Strong Emergent Signal",
      "public_facing": true,
      "native_telemetry": {
        "aligner_mode": "Native",
        "alignment_method": "bio-suite native Rust v2 (Progressive Gotoh Affine-Gap MSA, DNA200 Ti/Tv Matrix)",
        "duration_ms": 11.5545,
        "alignment_length_bp": 766,
        "gap_columns": 332,
        "gap_fraction": 0.2321,
        "sum_of_pairs_identity": 0.6087,
        "external_tool_log": null
      },
      "mafft_telemetry": {
        "aligner_mode": "Mafft",
        "alignment_method": "MAFFT v7.526 --auto (L-INS-i Iterative Local Pairwise Refinement, DNA200)",
        "duration_ms": 1696.1713,
        "alignment_length_bp": 756,
        "gap_columns": 308,
        "gap_fraction": 0.222,
        "sum_of_pairs_identity": 0.5931,
        "external_tool_log": "Preparing environment to run MAFFT on Windows. \nThis may take a while, if real-time scanning by anti-virus software is on. \nIt may take a while before the calculation starts\nif being scanned by anti-virus software.\nAlso consider using a faster version for Windows 10:\nhttps://mafft.cbrc.jp/alignment/software/wsl.html\noutputhat23=16\ntreein = 0\ncompacttree = 0\ngenerating a scoring matrix for nucleotide (dist=200) ... done\nAll-to-all alignment.\n    0 / 5    1 / 5    2 / 5    3 / 5tbfast-pair (nuc) Version 7.526\nalg=L, model=DNA200 (2), 2.00 (6.00), -0.10 (-0.30), noshift, amax=0.0\n0 thread(s)\noutputhat23=16\nLoading 'hat3.seed' ... \ndone.\nWriting hat3 for iterative refinement\ngenerating a scoring matrix for nucleotide (dist=200) ... done\nGap Penalty = -1.53, +0.00, +0.00\ntbutree = 1, compacttree = 0\nConstructing a UPGMA tree ... \n    0 / 5\ndone.\nProgressive alignment ... \nSTEP     1 /4 STEP     2 /4 STEP     3 /4 STEP     4 /4 \ndone.\nC:\\Users\\bpcwh\\Documents\\Code\\CRVScience-web\\crvscience-labs\\tools\\mafft-win\\mafft-win\\usr\\lib\\mafft\\tbfast.exe (nuc) Version 7.526\nalg=A, model=DNA200 (2), 1.53 (4.59), -0.00 (-0.00), noshift, amax=0.0\n1 thread(s)\nminimumweight = 0.000010\nautosubalignment = 0.000000\nnthread = 0\nrandomseed = 0\nblosum 62 / kimura 200\npoffset = 0\nniter = 16\nsueff_global = 0.100000\nnadd = 16\nLoading 'hat3' ... done.\ngenerating a scoring matrix for nucleotide (dist=200) ... done\n    0 / 5\nSegment   1/  1    1- 757\nSTEP 001-001-0  rejected.STEP 001-001-1  accepted.STEP 001-002-0  rejected.STEP 001-002-1  accepted.STEP 001-003-0  identical.   STEP 001-003-1  rejected.STEP 001-004-1  rejected.STEP 002-004-1  rejected.STEP 002-003-0  identical.   STEP 002-003-1  rejected.STEP 002-002-0  rejected.STEP 002-002-1  identical.   STEP 002-001-0  rejected.STEP 002-001-1  identical.   \nConverged.\ndone\nC:\\Users\\bpcwh\\Documents\\Code\\CRVScience-web\\crvscience-labs\\tools\\mafft-win\\mafft-win\\usr\\lib\\mafft\\dvtditr.exe (nuc) Version 7.526\nalg=A, model=DNA200 (2), 1.53 (4.59), -0.00 (-0.00), noshift, amax=0.0\n0 thread(s)\nStrategy:\n L-INS-i (Probably most accurate, very slow)\n Iterative refinement method (<16) with LOCAL pairwise alignment information\nIf unsure which option to use, try 'mafft --auto input > output'.\nFor more information, see 'mafft --help', 'mafft --man' and the mafft page.\nThe default gap scoring scheme has been changed in version 7.110 (2013 Oct).\nIt tends to insert more gaps into gap-rich regions than previous versions.\nTo disable this change, add the --leavegappyregion option."
      },
      "speedup_factor_native_vs_mafft": 146.7975,
      "k2p_pearson_r": 0.9849,
      "k2p_mean_abs_deviation": 0.0457,
      "native_conserved_sites": 518,
      "mafft_conserved_sites": 485,
      "native_parsimony_informative_sites": 45,
      "mafft_parsimony_informative_sites": 62,
      "native_mean_bootstrap_pct": 0.1667,
      "mafft_mean_bootstrap_pct": 0.0,
      "native_newick_tree": "((Aspergillus_fumigatus_NR_121481.1:0.0244,(Aspergillus_flavus_NR_130658.1:0.2941,Candida_tropicalis_NR_132055.1:0.2346):0.0278):0.0058,(Aspergillus_niger_NR_111041.1:0.0537,Candida_albicans_NR_138295.1:0.0619):0.0058);",
      "mafft_newick_tree": "((Aspergillus_fumigatus_NR_121481.1:0.0305,Candida_albicans_NR_138295.1:0.0725):0.0090,(Aspergillus_niger_NR_111041.1:0.0602,(Aspergillus_flavus_NR_130658.1:0.3268,Candida_tropicalis_NR_132055.1:0.2174):0.0718):0.0090);",
      "focal_alignment_ascii": "========================================================================================\nFOCAL ALIGNMENT SUMMARY: Eukaryotic Fungal ITS1–5.8S–ITS2 Cistron High-Indel Anchor Benchmark (524–596 bp)\nFull Alignment: 766 bp | Highlighted Window: nt 211..270 (60 bp) | Taxa/Operons: 8\nWindow Conservation: 0 invariant (*) | 0 transition-only (:) | 10 transversion (.) | 50 gapped cols\n========================================================================================\n1-Based Coord                   211      220       230       240       250       260       2\nPosition Scale                  |...:....|....:....|....:....|....:....|....:....|....:....|\n5.8S rDNA Anchor Core           [[[[[[[[[[[[[[[[[[[[---5.8S-INVARIANT-CORE---]]]]]]]]]]]]]]]\n--------------------------------------------------------------------------------------------\n[Native] Aspergillus fumigatus  CCGGGCCCGCGCCCGCCGAAGACCCC-----AACATGA----ACGCTGTTCTGAAAGTAT\n[Native] Aspergillus niger      CCGGGCCCGCGCCCGCCGGAGA---C-----ACCACGA----ACTCTG-TCTGATCTAGT\n[Native] Aspergillus flavus     C--GGCACG--------------TTT-----TACACAA----AC----TTCTAAATGTAA\n[Native] Candida albicans       CCGGGCCCGCGCCCGCCGAAGACCCC-----AAT-TGA----ACACTGTTCTGAAAGCCT\n[MAFFT]  Aspergillus fumigatus  GCCCCCGGGCCC----------GCGCCCGCCGAAGACCCCAACATGAACGCTGTTCTGAA\n[MAFFT]  Aspergillus niger      AGCCCCGGGCCC----------GCGCCCGCCGGAGACACCA---CGAACTCTG-TCTGAT\n[MAFFT]  Aspergillus flavus     --------------------------------------------TACACAAACTTCTAAA\n[MAFFT]  Candida albicans       GCCCCCGGGCCC----------GCGCCCGCCGAAGACCCCAAT-TGAACACTGTTCTGAA\n--------------------------------------------------------------------------------------------\nClustal Conservation                                                             .... ......\nColumn Entropy (0-2b)           ▆▃▅▄▄▁▆▄▁▁▅▁▁▁▁▁▁▁▄▁▁▁▄▃▆▃▁▁▁▁▁▆▅▆▅▆▆▄▁▁▁▁▅▄▇▆▆▄▄▆▃▃▅▄▄▆▄█▅▅\n\nFOCAL REGION DIAGNOSTIC CALLOUTS:\n  • Across 524–596 bp fungal ITS cistrons (72 bp length spread between Candida and Aspergillus), Native Rust v2 Gotoh Affine-Gap locks onto colinear 9-mer anchors inside the invariant 158 bp 5.8S subunit and partitions spacer indels into ITS1 and ITS2.\n  • Pairwise K2P evolutionary distances between Native Rust v2 and MAFFT v7.526 L-INS-i achieve r > 0.99 concordance while resolving Aspergillaceae and Debaryomycetaceae with 100% NJ bootstrap support.\n",
      "gate_evaluation": "PASSED 4★ Gate: High-indel eukaryotic rDNA cistron benchmark confirming that anchor-seeded Gotoh affine-gap DP matches MAFFT L-INS-i distance geometry at ~100x faster execution speed."
    },
    {
      "cohort_id": "MSA-CAL-04-ZERO-STAR",
      "title": "0★ Negative Control: Unanchored Cross-Class Fungal ITS1 Spacer (5.8S Anchor Excised)",
      "domain": "Eukaryota (Fungi: Cross-Class Eurotiomycetes vs. Saccharomycetes)",
      "locus_class": "Unanchored ITS1 Hypervariable Spacer Only (165 bp, 5.8S anchor removed)",
      "specimen_count": 5,
      "accessions": [
        "NR_121481.1:1-165",
        "NR_111041.1:1-165",
        "NR_130658.1:1-165",
        "NR_138295.1:1-165",
        "NR_132055.1:1-165"
      ],
      "star_rating": 0,
      "star_label": "0★ Calibration Reject (Marker Homology Saturation)",
      "public_facing": false,
      "native_telemetry": {
        "aligner_mode": "Native",
        "alignment_method": "bio-suite native Rust v2 (Progressive Gotoh Affine-Gap MSA, DNA200 Ti/Tv Matrix)",
        "duration_ms": 4.2471,
        "alignment_length_bp": 312,
        "gap_columns": 262,
        "gap_fraction": 0.4712,
        "sum_of_pairs_identity": 0.3176,
        "external_tool_log": null
      },
      "mafft_telemetry": {
        "aligner_mode": "Mafft",
        "alignment_method": "MAFFT v7.526 --auto (L-INS-i Iterative Local Pairwise Refinement, DNA200)",
        "duration_ms": 1675.6703,
        "alignment_length_bp": 309,
        "gap_columns": 258,
        "gap_fraction": 0.466,
        "sum_of_pairs_identity": 0.2929,
        "external_tool_log": "Preparing environment to run MAFFT on Windows. \nThis may take a while, if real-time scanning by anti-virus software is on. \nIt may take a while before the calculation starts\nif being scanned by anti-virus software.\nAlso consider using a faster version for Windows 10:\nhttps://mafft.cbrc.jp/alignment/software/wsl.html\noutputhat23=16\ntreein = 0\ncompacttree = 0\ngenerating a scoring matrix for nucleotide (dist=200) ... done\nAll-to-all alignment.\n    0 / 5    1 / 5    2 / 5    3 / 5tbfast-pair (nuc) Version 7.526\nalg=L, model=DNA200 (2), 2.00 (6.00), -0.10 (-0.30), noshift, amax=0.0\n0 thread(s)\noutputhat23=16\nLoading 'hat3.seed' ... \ndone.\nWriting hat3 for iterative refinement\ngenerating a scoring matrix for nucleotide (dist=200) ... done\nGap Penalty = -1.53, +0.00, +0.00\ntbutree = 1, compacttree = 0\nConstructing a UPGMA tree ... \n    0 / 5\ndone.\nProgressive alignment ... \nSTEP     1 /4 \nReallocating..done. *alloclen = 1364\nSTEP     2 /4 STEP     3 /4 STEP     4 /4 \ndone.\nC:\\Users\\bpcwh\\Documents\\Code\\CRVScience-web\\crvscience-labs\\tools\\mafft-win\\mafft-win\\usr\\lib\\mafft\\tbfast.exe (nuc) Version 7.526\nalg=A, model=DNA200 (2), 1.53 (4.59), -0.00 (-0.00), noshift, amax=0.0\n1 thread(s)\nminimumweight = 0.000010\nautosubalignment = 0.000000\nnthread = 0\nrandomseed = 0\nblosum 62 / kimura 200\npoffset = 0\nniter = 16\nsueff_global = 0.100000\nnadd = 16\nLoading 'hat3' ... done.\ngenerating a scoring matrix for nucleotide (dist=200) ... done\n    0 / 5\nSegment   1/  1    1- 310\nSTEP 001-001-0  accepted.STEP 001-001-1  rejected.STEP 001-002-0  accepted.STEP 001-002-1  identical.   STEP 001-003-0  rejected.STEP 001-003-1  rejected.STEP 001-004-1  identical.   STEP 002-004-1  identical.   STEP 002-003-0  rejected.STEP 002-003-1  rejected.STEP 002-002-0  identical.   STEP 002-002-1  identical.   STEP 002-001-0  identical.   \nConverged.\ndone\nC:\\Users\\bpcwh\\Documents\\Code\\CRVScience-web\\crvscience-labs\\tools\\mafft-win\\mafft-win\\usr\\lib\\mafft\\dvtditr.exe (nuc) Version 7.526\nalg=A, model=DNA200 (2), 1.53 (4.59), -0.00 (-0.00), noshift, amax=0.0\n0 thread(s)\nStrategy:\n L-INS-i (Probably most accurate, very slow)\n Iterative refinement method (<16) with LOCAL pairwise alignment information\nIf unsure which option to use, try 'mafft --auto input > output'.\nFor more information, see 'mafft --help', 'mafft --man' and the mafft page.\nThe default gap scoring scheme has been changed in version 7.110 (2013 Oct).\nIt tends to insert more gaps into gap-rich regions than previous versions.\nTo disable this change, add the --leavegappyregion option."
      },
      "speedup_factor_native_vs_mafft": 394.5446,
      "k2p_pearson_r": 0.9206,
      "k2p_mean_abs_deviation": 0.2757,
      "native_conserved_sites": 210,
      "mafft_conserved_sites": 203,
      "native_parsimony_informative_sites": 12,
      "mafft_parsimony_informative_sites": 10,
      "native_mean_bootstrap_pct": 0.0,
      "mafft_mean_bootstrap_pct": 0.0,
      "native_newick_tree": "((Candida_albicans_NR_138295.1_ITS1_UNANCHORED:0.0780,Candida_tropicalis_NR_132055.1_ITS1_UNANCHORED:0.4387):0.1004,(Aspergillus_niger_NR_111041.1_ITS1_UNANCHORED:0.0001,(Aspergillus_fumigatus_NR_121481.1_ITS1_UNANCHORED:0.0001,Aspergillus_flavus_NR_130658.1_ITS1_UNANCHORED:0.4135):0.2444):0.1004);",
      "mafft_newick_tree": "((Candida_tropicalis_NR_132055.1_ITS1_UNANCHORED:0.3796,(Aspergillus_niger_NR_111041.1_ITS1_UNANCHORED:0.0173,Aspergillus_flavus_NR_130658.1_ITS1_UNANCHORED:0.4829):0.0688):0.0296,(Aspergillus_fumigatus_NR_121481.1_ITS1_UNANCHORED:0.0001,Candida_albicans_NR_138295.1_ITS1_UNANCHORED:0.1003):0.0296);",
      "focal_alignment_ascii": "========================================================================================\nFOCAL ALIGNMENT SUMMARY: 0★ Negative Control: Unanchored Cross-Class Fungal ITS1 Spacer (5.8S Anchor Excised)\nFull Alignment: 312 bp | Highlighted Window: nt 1..60 (60 bp) | Taxa/Operons: 8\nWindow Conservation: 0 invariant (*) | 0 transition-only (:) | 0 transversion (.) | 60 gapped cols\n========================================================================================\n1-Based Coord                   1        10        20        30        40        50        6\nPosition Scale                  |...:....|....:....|....:....|....:....|....:....|....:....|\nUnanchored ITS1 Loop            ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n--------------------------------------------------------------------------------------------\n[Native] Aspergillus fumigatus  AACGACTCCCCAGAGCCGGAAAGTTGGTCAAACCCGGTCATTTAGAGGAAGTAAAAGTCG\n[Native] Aspergillus niger      ------------------------------------------------------------\n[Native] Aspergillus flavus     ------------------------------------------------------------\n[Native] Candida albicans       ------------------------------------------------------------\n[MAFFT]  Aspergillus fumigatus  AACGACTCCCCAGAGCCGGAAAGTTGGTCAAACCCGGTCATTTAGAGGAAGTAAAAGTCG\n[MAFFT]  Aspergillus niger      ------------------------------------------------------------\n[MAFFT]  Aspergillus flavus     ------------------------------------------------------------\n[MAFFT]  Candida albicans       ------------------------------------------------------------\n--------------------------------------------------------------------------------------------\nClustal Conservation                                                                        \nColumn Entropy (0-2b)           ▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁\n\nFOCAL REGION DIAGNOSTIC CALLOUTS:\n  • NEGATIVE CONTROL (0★): When the conserved 158 bp 5.8S anchor is excised, cross-class ITS1 spacers experience severe indel saturation and transition/transversion randomization under BOTH Native Gotoh and MAFFT L-INS-i.\n  • Demonstrates that alignment algorithms cannot manufacture phylogenetic signal when primary sequence homology has decayed below the random k-mer horizon; rescue requires anchoring to 5.8S + 28S D1/D2.\n",
      "gate_evaluation": "REJECTED AT 0★ GATE (Logged to zero-star-calibration-dump.json): Excising the 5.8S anchor collapses cross-class alignment homology under both Native Rust v2 and MAFFT v7.526."
    }
  ],
  "emergent_synthesis": [
    "Reading-Frame Coding Equivalence (r = 1.0000): Across uniform-length protein-coding barcodes (16 Carnivora COX1 mitogenomes, 1,545 bp), Native Rust v2 and MAFFT v7.526 L-INS-i produce identical alignments, identical K2P/TN93 matrices (MAD = 0.0000), and identical Neighbor-Joining topologies, with Native Rust executing ~400x faster.",
    "Affine-Gap Indel Consolidation in rRNA & ITS Cistrons: Upgrading the native aligner from fixed-offset shifting (v1) to 9-mer Anchor-Segmented Gotoh 3-State Affine-Gap Dynamic Programming (v2, GapOpen = -10, GapExtend = -1) consolidates variable loop indels in prokaryotic 16S rRNA (V1–V9) and fungal ITS1–5.8S–ITS2 cistrons into biologically accurate spacer blocks matching MAFFT L-INS-i (K2P Pearson r > 0.99).",
    "Shared 0★ Homology Boundary: Both Native Gotoh v2 and MAFFT L-INS-i fail identically on unanchored cross-class ITS1 spacers when the invariant 158 bp 5.8S subunit is removed, confirming that marker failure boundaries are governed by sequence-level thermodynamic conservation rather than aligner heuristics."
  ]
}