{ "schema_version": 2, "experiment_date": "2026-08-01", "status": "research-only FASTQ-path candidate-recovery follow-up", "privacy": "Pseudonymized aggregate: exact per-control counts may fingerprint a known library; this is not anonymized genomic data.", "assembly": "GRCh37/hg19", "question": "Should synthetic BAM alignment tags be sampled, or should fusion molecules be generated as FASTQ and passed through alignment?", "answer": "Use FASTQ followed by the production aligner. Do not independently copy or sample AS, XS, MAPQ, CIGAR, SA, or alignment flags.", "input_inventory": { "project_fastq_files_found_before_followup": 0, "expected_original_fastq_basenames_searched": 14, "expected_original_fastq_basenames_found": 0, "synchronized_fastq_family_files_examined": 88, "full_hg19_fasta_available": true, "full_hg19_contigs": 93, "bam_primary_contigs_matching_reference_lengths": "25/25", "breakpoint_panel_tiles_matching_full_reference": "4/4", "original_alignment_program": "BWA-MEM 0.7.12-r1039", "original_alignment_mode": "bwa mem -M", "pinned_aligner_runtime_and_index_available_locally": false }, "design": { "controls": 7, "eml4_alk_controls": 5, "cd74_ros1_controls": 2, "background_source": "complete primary pairs reconstructed from locked, duplicate-removed BAMs in sequencer orientation", "target_window_bp": 5, "target_definition": "ordered gene pair, chromosome, strand orientation, and both breakpoints within +/-5 bp", "matched_background_and_spiked_fastq_called": true, "caller_scope": "FusionSieve direct FASTQ search against targeted panel tiles; not whole-hg19 realignment", "truth_reconciled_after_calling": true }, "final_caller_configuration": { "strata": [ { "controls": [ "C01", "C02", "C03", "C06", "C07" ], "read_length": 151, "min_anchor": 25, "min_align_identity": 0.9, "scientific_config_sha256": "234f3d82e6d4dbff23936d79ab201b14a460934639db73a719be5174f8192747" }, { "controls": [ "C04", "C05" ], "read_length": 251, "min_anchor": 25, "min_align_identity": 0.91, "scientific_config_sha256": "edfe00088774370881037127014b32e81b8ffceb238320e529e202e4a95e4418" } ], "selection_rule": "Modal read length from each reconstructed background; min_anchor 25; minimum alignment identity 0.90 for 151-bp controls and 0.91 for 251-bp controls to satisfy FusionSieve's triage guard.", "audit_correction": "An independent pre-publication audit found the preliminary 150-bp default in all runs. All seven controls were rerun with input-dependent final configs. C04/C05 all-candidate counts changed materially, while target-window recovery did not.", "preliminary_to_final_candidate_counts_251bp": { "C04": { "baseline": "1149 -> 866", "spiked": "1150 -> 867" }, "C05": { "baseline": "1089 -> 755", "spiked": "1090 -> 756" } }, "target_specific_retuning": false, "automatic_reporting_enabled": false, "validated_model_supplied": false, "compatible_panel_of_normals_supplied": false }, "primary_results": { "target_candidates_recovered_within_5bp": 7, "controls_tested": 7, "baseline_target_window_matches": 0, "spiked_target_window_matches": 7, "ambiguous_target_window_matches": 0, "max_absolute_breakpoint_error_bp": 2, "recovered_candidates_with_no_hard_filters": 7, "review_tier_target_candidates": 7, "reportable_pass_or_high_targets": 0, "interpretation": "7/7 planted junctions were recovered as correctly oriented +/-5-bp REVIEW candidates; 0/7 were automatic PASS/HIGH reportable calls." }, "generation": { "bam_records_examined": 3546125, "secondary_or_supplementary_records_excluded": 13205, "unmatched_primary_ends_excluded": 1462, "other_extraction_exclusions": 0, "background_pairs": 1765729, "pairs_added": 553, "split_fragments_requested": 420, "split_fragments_planted": 420, "split_read_ends_planted": 551, "spanning_pairs_requested": 140, "spanning_pairs_planted": 133, "spanning_shortfall": 7 }, "caller_evidence": { "split_reads": 470, "unique_split_reads": 385, "spanning_pairs": 121, "unique_spanning_pairs": 121, "total_support": 506, "fusion_support_fraction_interpretation": "FusionSieve evidence proxy, not tumor allele fraction, molecular VAF, or validated molecule count." }, "prior_direct_bam_realism_audit": { "relationship_to_followup": "Imported evidence from the prior direct-BAM spike experiment on the same seven controls; this FASTQ follow-up produced no BAM.", "native_comparator_definition": "Non-planted records whose 1-based alignment start was strictly less than 2,500 bp from either planted breakpoint, pooled across the seven BAMs.", "nearby_native_records": 100287, "planted_records": 1681, "tag_presence": { "AS": { "native": 100287, "native_percent": 100.0, "planted": 0, "planted_percent": 0.0 }, "XS": { "native": 100287, "native_percent": 100.0, "planted": 0, "planted_percent": 0.0 }, "NM": { "native": 100241, "native_percent": 99.9541, "planted": 1681, "planted_percent": 100.0 }, "MD": { "native": 100238, "native_percent": 99.9511, "planted": 1681, "planted_percent": 100.0 }, "SA": { "native": 120, "native_percent": 0.1197, "planted": 1134, "planted_percent": 67.46 } }, "mapq_median": { "native": 60, "planted": 60 }, "mean_base_quality_median": { "native": 35.2, "planted": 34.8 }, "nm_median": { "native": 0, "planted": 0 }, "native_alignment_structure": { "records_examined": 3546125, "secondary": 13205, "supplementary": 0, "hard_clipped": 13205 }, "planted_alignment_structure": { "records_examined": 1681, "secondary": 0, "supplementary": 567, "hard_clipped": 0, "soft_clipped": 1134 }, "binary_tag_type_fingerprint": "Planted NM/MQ used generic integer storage while native BAMs used compact c/C integer encodings.", "interpretation": "In these BAMs, the original BWA -M processing represented split hits as secondary hard-clipped records, whereas the direct BAM spike used supplementary soft-clipped records. Sampled AS/XS values cannot repair this coupled structural mismatch. This is an observation in this dataset, not a universal claim about BWA." }, "integrity": { "background_extraction_files_verified": 14, "spikeforge_input_output_files_verified": 28, "caller_input_integrity_manifests_verified": 14, "caller_output_json_verified": 14, "baseline_spiked_scientific_configs_matched": 7, "baseline_spiked_runtime_panels_matched": 7, "spikeforge_manifest_designs_verified": 7, "spikeforge_truth_artifacts_verified": 21, "spikeforge_runtime_package_files_verified": 20, "source_binding_scope": "Current manifests verify BAM-derived FASTQ files onward. The historical extraction summaries did not record source BAM fingerprints, so they do not independently prove the source-BAM-to-FASTQ derivation link." }, "provenance": { "spikeforge_version": "1.1.0rc2", "spikeforge_release_zip_name": "spikeforge-1.1.0rc2-research-preview.zip", "spikeforge_release_zip_bytes": 374774, "spikeforge_release_zip_sha256": "8dec28c1234503d5fc2ac3658f745e89bf324c891f420db4b9f3095e2ae0f5df", "spikeforge_runtime_package_sha256": "2934899d283e85093a2acd1ec5c0420de7372ee82b8dac78bb9cbe87aabe1400", "fusionsieve_versions": [ "1.1.0.dev0" ], "fusionsieve_source_sha256": [ "8d9ec7e47445917fda75422e9e0152ead8e0b9c08a34bd3703d21de80b0be35c" ], "panel_fasta_sha256": [ "b7a5a05d5cf5d649fd74713e52865b9a106e5a548af834c6ac8e400c5c9ff0ed" ], "panel_json_sha256": [ "c32bfdace27a220b53652918cda0111ca0582dc00a87c966c53f6bf4a4fbff09" ], "scientific_config_strata": [ { "controls": [ "C01", "C02", "C03", "C06", "C07" ], "read_length": 151, "min_anchor": 25, "min_align_identity": 0.9, "scientific_config_sha256": "234f3d82e6d4dbff23936d79ab201b14a460934639db73a719be5174f8192747" }, { "controls": [ "C04", "C05" ], "read_length": 251, "min_anchor": 25, "min_align_identity": 0.91, "scientific_config_sha256": "edfe00088774370881037127014b32e81b8ffceb238320e529e202e4a95e4418" } ] }, "controls": [ { "control": "C01", "cohort": "historical", "fusion": "EML4-ALK", "seed": 20260801, "bam_records_examined": 372689, "secondary_or_supplementary_records_excluded": 904, "unmatched_primary_ends_excluded": 47, "other_extraction_exclusions": 0, "background_pairs": 185869, "pairs_added": 80, "split_fragments_planted": 60, "split_read_ends_planted": 71, "spanning_pairs_planted": 20, "baseline_all_candidates": 3713, "spiked_all_candidates": 3714, "baseline_gene_pair_candidates": 29, "spiked_gene_pair_candidates": 30, "baseline_target_window_matches": 0, "spiked_target_window_matches": 1, "target_window_match_ambiguous": false, "target_candidate_recovered": true, "target_reportable": false, "nearest_gene_pair_distance_sum_bp": 4, "left_error_bp": -2, "right_error_bp": 2, "tier": "REVIEW", "score": 1.0, "hard_filters": [], "soft_filters": [ "low_depth", "auto_reporting_disabled", "unvalidated_scorer", "missing_pon", "unverified_duplicate_processing" ], "split_reads": 55, "split_reads_unique": 51, "spanning_pairs": 20, "spanning_pairs_unique": 20, "total_support": 71, "fusion_support_fraction": 0.122414, "reportable_calls_baseline": 0, "reportable_calls_spiked": 0, "caller_read_length": 151, "caller_min_anchor": 25, "caller_min_align_identity": 0.9, "scientific_config_sha256": "234f3d82e6d4dbff23936d79ab201b14a460934639db73a719be5174f8192747", "fusionsieve_version": "1.1.0.dev0", "fusionsieve_source_sha256": "8d9ec7e47445917fda75422e9e0152ead8e0b9c08a34bd3703d21de80b0be35c", "panel_fasta_sha256": "b7a5a05d5cf5d649fd74713e52865b9a106e5a548af834c6ac8e400c5c9ff0ed", "panel_json_sha256": "c32bfdace27a220b53652918cda0111ca0582dc00a87c966c53f6bf4a4fbff09", "spikeforge_version": "1.1.0rc2", "spikeforge_release_zip_sha256": "8dec28c1234503d5fc2ac3658f745e89bf324c891f420db4b9f3095e2ae0f5df", "spikeforge_runtime_package_sha256": "2934899d283e85093a2acd1ec5c0420de7372ee82b8dac78bb9cbe87aabe1400" }, { "control": "C02", "cohort": "historical", "fusion": "EML4-ALK", "seed": 20260802, "bam_records_examined": 452774, "secondary_or_supplementary_records_excluded": 940, "unmatched_primary_ends_excluded": 30, "other_extraction_exclusions": 0, "background_pairs": 225902, "pairs_added": 80, "split_fragments_planted": 60, "split_read_ends_planted": 78, "spanning_pairs_planted": 20, "baseline_all_candidates": 4629, "spiked_all_candidates": 4630, "baseline_gene_pair_candidates": 33, "spiked_gene_pair_candidates": 34, "baseline_target_window_matches": 0, "spiked_target_window_matches": 1, "target_window_match_ambiguous": false, "target_candidate_recovered": true, "target_reportable": false, "nearest_gene_pair_distance_sum_bp": 4, "left_error_bp": -2, "right_error_bp": 2, "tier": "REVIEW", "score": 1.0, "hard_filters": [], "soft_filters": [ "low_depth", "auto_reporting_disabled", "unvalidated_scorer", "missing_pon", "unverified_duplicate_processing" ], "split_reads": 66, "split_reads_unique": 55, "spanning_pairs": 18, "spanning_pairs_unique": 18, "total_support": 73, "fusion_support_fraction": 0.130318, "reportable_calls_baseline": 0, "reportable_calls_spiked": 0, "caller_read_length": 151, "caller_min_anchor": 25, "caller_min_align_identity": 0.9, "scientific_config_sha256": "234f3d82e6d4dbff23936d79ab201b14a460934639db73a719be5174f8192747", "fusionsieve_version": "1.1.0.dev0", "fusionsieve_source_sha256": "8d9ec7e47445917fda75422e9e0152ead8e0b9c08a34bd3703d21de80b0be35c", "panel_fasta_sha256": "b7a5a05d5cf5d649fd74713e52865b9a106e5a548af834c6ac8e400c5c9ff0ed", "panel_json_sha256": "c32bfdace27a220b53652918cda0111ca0582dc00a87c966c53f6bf4a4fbff09", "spikeforge_version": "1.1.0rc2", "spikeforge_release_zip_sha256": "8dec28c1234503d5fc2ac3658f745e89bf324c891f420db4b9f3095e2ae0f5df", "spikeforge_runtime_package_sha256": "2934899d283e85093a2acd1ec5c0420de7372ee82b8dac78bb9cbe87aabe1400" }, { "control": "C03", "cohort": "historical", "fusion": "EML4-ALK", "seed": 20260803, "bam_records_examined": 431742, "secondary_or_supplementary_records_excluded": 971, "unmatched_primary_ends_excluded": 33, "other_extraction_exclusions": 0, "background_pairs": 215369, "pairs_added": 80, "split_fragments_planted": 60, "split_read_ends_planted": 68, "spanning_pairs_planted": 20, "baseline_all_candidates": 4660, "spiked_all_candidates": 4661, "baseline_gene_pair_candidates": 31, "spiked_gene_pair_candidates": 32, "baseline_target_window_matches": 0, "spiked_target_window_matches": 1, "target_window_match_ambiguous": false, "target_candidate_recovered": true, "target_reportable": false, "nearest_gene_pair_distance_sum_bp": 4, "left_error_bp": -2, 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"unvalidated_scorer", "missing_pon", "unverified_duplicate_processing" ], "split_reads": 64, "split_reads_unique": 59, "spanning_pairs": 20, "spanning_pairs_unique": 20, "total_support": 79, "fusion_support_fraction": 0.578289, "reportable_calls_baseline": 0, "reportable_calls_spiked": 0, "caller_read_length": 251, "caller_min_anchor": 25, "caller_min_align_identity": 0.91, "scientific_config_sha256": "edfe00088774370881037127014b32e81b8ffceb238320e529e202e4a95e4418", "fusionsieve_version": "1.1.0.dev0", "fusionsieve_source_sha256": "8d9ec7e47445917fda75422e9e0152ead8e0b9c08a34bd3703d21de80b0be35c", "panel_fasta_sha256": "b7a5a05d5cf5d649fd74713e52865b9a106e5a548af834c6ac8e400c5c9ff0ed", "panel_json_sha256": "c32bfdace27a220b53652918cda0111ca0582dc00a87c966c53f6bf4a4fbff09", "spikeforge_version": "1.1.0rc2", "spikeforge_release_zip_sha256": "8dec28c1234503d5fc2ac3658f745e89bf324c891f420db4b9f3095e2ae0f5df", "spikeforge_runtime_package_sha256": 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"unverified_duplicate_processing" ], "split_reads": 81, "split_reads_unique": 54, "spanning_pairs": 13, "spanning_pairs_unique": 13, "total_support": 67, "fusion_support_fraction": 0.097028, "reportable_calls_baseline": 0, "reportable_calls_spiked": 0, "caller_read_length": 151, "caller_min_anchor": 25, "caller_min_align_identity": 0.9, "scientific_config_sha256": "234f3d82e6d4dbff23936d79ab201b14a460934639db73a719be5174f8192747", "fusionsieve_version": "1.1.0.dev0", "fusionsieve_source_sha256": "8d9ec7e47445917fda75422e9e0152ead8e0b9c08a34bd3703d21de80b0be35c", "panel_fasta_sha256": "b7a5a05d5cf5d649fd74713e52865b9a106e5a548af834c6ac8e400c5c9ff0ed", "panel_json_sha256": "c32bfdace27a220b53652918cda0111ca0582dc00a87c966c53f6bf4a4fbff09", "spikeforge_version": "1.1.0rc2", "spikeforge_release_zip_sha256": "8dec28c1234503d5fc2ac3658f745e89bf324c891f420db4b9f3095e2ae0f5df", "spikeforge_runtime_package_sha256": "2934899d283e85093a2acd1ec5c0420de7372ee82b8dac78bb9cbe87aabe1400" }, { "control": "C07", "cohort": "historical", "fusion": "CD74-ROS1", "seed": 20260807, "bam_records_examined": 889282, "secondary_or_supplementary_records_excluded": 4337, "unmatched_primary_ends_excluded": 713, "other_extraction_exclusions": 0, "background_pairs": 442116, "pairs_added": 75, "split_fragments_planted": 60, "split_read_ends_planted": 98, "spanning_pairs_planted": 15, "baseline_all_candidates": 9078, "spiked_all_candidates": 9079, "baseline_gene_pair_candidates": 1, "spiked_gene_pair_candidates": 2, "baseline_target_window_matches": 0, "spiked_target_window_matches": 1, "target_window_match_ambiguous": false, "target_candidate_recovered": true, "target_reportable": false, "nearest_gene_pair_distance_sum_bp": 4, "left_error_bp": 2, "right_error_bp": 2, "tier": "REVIEW", "score": 1.0, "hard_filters": [], "soft_filters": [ "auto_reporting_disabled", "unvalidated_scorer", "missing_pon", "unverified_duplicate_processing" ], "split_reads": 80, "split_reads_unique": 52, "spanning_pairs": 11, "spanning_pairs_unique": 11, "total_support": 63, "fusion_support_fraction": 0.080163, "reportable_calls_baseline": 0, "reportable_calls_spiked": 0, "caller_read_length": 151, "caller_min_anchor": 25, "caller_min_align_identity": 0.9, "scientific_config_sha256": "234f3d82e6d4dbff23936d79ab201b14a460934639db73a719be5174f8192747", "fusionsieve_version": "1.1.0.dev0", "fusionsieve_source_sha256": "8d9ec7e47445917fda75422e9e0152ead8e0b9c08a34bd3703d21de80b0be35c", "panel_fasta_sha256": "b7a5a05d5cf5d649fd74713e52865b9a106e5a548af834c6ac8e400c5c9ff0ed", "panel_json_sha256": "c32bfdace27a220b53652918cda0111ca0582dc00a87c966c53f6bf4a4fbff09", "spikeforge_version": "1.1.0rc2", "spikeforge_release_zip_sha256": "8dec28c1234503d5fc2ac3658f745e89bf324c891f420db4b9f3095e2ae0f5df", "spikeforge_runtime_package_sha256": "2934899d283e85093a2acd1ec5c0420de7372ee82b8dac78bb9cbe87aabe1400" } ], "limitations": [ "The original raw FASTQs were not available.", "BAM-derived FASTQs omit duplicate-removed reads and retain post-BQSR qualities.", "The direct FASTQ caller searched targeted panel tiles, not whole hg19, so genome-wide alternative-hit competition was absent.", "This run does not validate BWA-derived AS/XS/MAPQ/CIGAR or primary-secondary-supplementary structure.", "The extraction workflow located locked BAMs by size and SHA-256, but its historical extraction summaries did not persist a cryptographic source-BAM-to-derived-FASTQ binding.", "All target recoveries were REVIEW candidates; automatic reporting was intentionally disabled and no validated scorer or compatible panel of normals was supplied.", "Per-control support and fusion_support_fraction are evidence proxies, not tumor VAF or validated molecule counts.", "This experiment does not establish clinical performance or treatment utility." ] }