{ "$schema": "https://json-schema.org/draft/2020-12/schema", "$id": "https://scischema.org/schemas/biology/pcr/1.0.0/master-schema.json", "version": "1.0.0", "title": "Polymerase Chain Reaction (PCR) Process Schema", "description": "Comprehensive schema for PCR processes including standard endpoint PCR, real-time qPCR, RT-qPCR, single-cell PCR, PCR-ASO, and droplet digital PCR (ddPCR). Covers reagents, thermal cycling, detection methods, curve analysis, and performance metrics.", "type": "object", "$defs": { "reagent": { "type": "object", "description": "Standardized structure for reagent quantities and provenance.", "properties": { "concentration": { "type": "number", "description": "Concentration in appropriate units (e.g., µM for primers, U/µL for enzymes, mM for dNTPs, ng/µL for DNA).", "minimum": 0 }, "volume": { "type": "number", "description": "Volume added to the reaction in µL.", "minimum": 0 }, "units": { "type": "string", "description": "Concentration units (e.g., µM, U/µL, mM, ng/µL, copies/µL)." }, "manufacturer": { "type": "string", "description": "Manufacturer or vendor of the reagent." }, "catalog_number": { "type": "string", "description": "Manufacturer catalog or part number for the reagent." }, "lot_number": { "type": "string", "description": "Lot number for traceability." } }, "required": [ "concentration", "volume" ] } }, "properties": { "experiment_metadata": { "type": "object", "description": "Administrative and contextual metadata for the experiment.", "properties": { "experiment_id": { "type": "string", "description": "Unique identifier for the PCR experiment." }, "experiment_date": { "type": "string", "description": "Date when the experiment was conducted.", "format": "date" }, "operator": { "type": "string", "description": "Name or identifier of the person conducting the experiment." }, "lab_notebook_reference": { "type": "string", "description": "Reference to the lab notebook or electronic record." }, "replicates": { "type": "integer", "description": "Number of replicates for the experiment.", "minimum": 1 }, "notes": { "type": "string", "description": "Additional notes or observations about the experiment." } }, "required": [ "experiment_id" ] }, "sample_preparation": { "type": "object", "description": "Sample origin, type, extraction, and quality.", "properties": { "sample_type": { "type": "string", "description": "Type of sample used.", "enum": [ "tissue", "blood", "cell-free DNA", "circulating tumor cells", "FFPE", "RNA", "single-cell", "other" ] }, "sample_source": { "type": "string", "description": "Biological source of the sample (e.g., plasma, serum, urine, CSF, sputum, pleural effusion, ascites, stool, fresh tissue, exosomes)." }, "extraction_method": { "type": "string", "description": "Nucleic acid extraction method (e.g., column-based, magnetic beads, phenol-chloroform, TRIzol, direct lysis)." }, "sample_purity": { "type": "string", "description": "Description of sample purity." }, "sample_degradation": { "type": "string", "description": "Assessment of sample degradation.", "enum": [ "intact", "partially_degraded", "highly_degraded" ] }, "archival_sample": { "type": "boolean", "description": "Indicates if the sample is archival." }, "reverse_transcription": { "type": "object", "description": "Parameters for RNA reverse transcription (RT-PCR).", "properties": { "performed": { "type": "boolean", "description": "Indicates if reverse transcription was performed." }, "enzyme": { "type": "string", "description": "Type of reverse transcriptase enzyme used.", "enum": [ "M-MLV", "SuperScript III", "AMV", "other" ] }, "primer_type": { "type": "string", "description": "Type of primer used for reverse transcription.", "enum": [ "oligo(dT)", "random hexamers", "gene-specific primers", "other" ] }, "temperature": { "type": "number", "description": "Temperature for reverse transcription in °C.", "minimum": 0 }, "time": { "type": "number", "minimum": 0, "description": "Duration in minutes." }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "performed" ] }, "single_cell_pcr": { "type": "object", "description": "Parameters specific to single-cell PCR.", "properties": { "isolation_method": { "type": "string", "description": "Cell isolation method (e.g., micromanipulation, laser capture microdissection, flow cytometry, microfluidics)." }, "amplification_bias": { "type": "string", "description": "Description of bias in amplification of low-, medium-, and high-abundance transcripts." }, "preamplification": { "type": "boolean", "description": "Indicates if preamplification was performed." }, "preamplification_method": { "type": "string", "description": "Method used for preamplification." } } } } }, "reaction_components": { "type": "object", "description": "All reagents used in the PCR reaction.", "properties": { "template_dna": { "type": "object", "description": "The DNA sample containing the target sequence to be amplified. Can be genomic DNA, plasmid DNA, or cDNA. Includes reagent details for the template DNA.", "properties": { "type": { "type": "string", "description": "Type of template.", "enum": [ "genomic DNA", "plasmid DNA", "cDNA", "synthetic DNA", "cell lysate", "single-cell DNA", "cfDNA", "ctDNA", "RNA", "other" ] }, "source": { "type": "string", "description": "Source of the template DNA." }, "initial_quantity": { "type": "number", "description": "Initial quantity of template in ng, pg, or number of cells.", "minimum": 0 }, "purity": { "type": "string", "description": "Description of template purity." }, "degradation_status": { "type": "string", "description": "Assessment of template degradation." }, "contamination": { "type": "string", "description": "Description of potential contaminants." }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "type", "reagent_details" ] }, "primers": { "type": "object", "description": "Forward and reverse primers flanking the target region.", "properties": { "forward_primer": { "type": "object", "description": "The forward primer sequence (5' to 3') that binds to the 3' end of the template strand.", "properties": { "sequence": { "type": "string", "description": "Nucleotide sequence of the forward primer.", "pattern": "^[ATCGatcg]+$" }, "length": { "type": "integer", "description": "Length of the forward primer in nucleotides (nt).", "minimum": 1 }, "tm": { "type": "number", "description": "Melting temperature (Tm) of the forward primer in °C.", "minimum": 0 }, "gc_content": { "type": "number", "description": "GC content of the primer as a percentage.", "minimum": 0, "maximum": 100 }, "modifications": { "type": "string", "description": "Chemical modifications." }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "sequence", "length", "reagent_details" ] }, "reverse_primer": { "type": "object", "description": "The reverse primer sequence (5' to 3') that binds to the 3' end of the complementary template strand.", "properties": { "sequence": { "type": "string", "description": "Nucleotide sequence of the reverse primer.", "pattern": "^[ATCGatcg]+$" }, "length": { "type": "integer", "description": "Length of the reverse primer in nucleotides (nt).", "minimum": 1 }, "tm": { "type": "number", "description": "Melting temperature (Tm) of the reverse primer in °C.", "minimum": 0 }, "gc_content": { "type": "number", "description": "GC content of the primer as a percentage.", "minimum": 0, "maximum": 100 }, "modifications": { "type": "string", "description": "Chemical modifications." }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "sequence", "length", "reagent_details" ] }, "nested_primers": { "type": "object", "description": "Optional nested primer pair for second-stage amplification.", "properties": { "forward_primer": { "type": "object", "description": "Forward primer used in the second (nested) round of amplification.", "properties": { "sequence": { "type": "string", "description": "Nucleotide sequence of the forward nested primer (5' to 3').", "pattern": "^[ATCGatcg]+$" }, "length": { "type": "integer", "description": "Length of the forward nested primer in bases.", "minimum": 1 }, "tm": { "type": "number", "description": "Melting temperature (Tm) of the forward nested primer in °C.", "minimum": 0 }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "sequence", "length", "reagent_details" ] }, "reverse_primer": { "type": "object", "description": "Reverse primer used in the second (nested) round of amplification.", "properties": { "sequence": { "type": "string", "description": "Nucleotide sequence of the reverse nested primer (5' to 3').", "pattern": "^[ATCGatcg]+$" }, "length": { "type": "integer", "description": "Length of the reverse nested primer in bases.", "minimum": 1 }, "tm": { "type": "number", "description": "Melting temperature (Tm) of the reverse nested primer in °C.", "minimum": 0 }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "sequence", "length", "reagent_details" ] } } }, "allele_specific_primers": { "type": "array", "description": "Allele-specific primers for SNP or mutation detection.", "items": { "type": "object", "properties": { "name": { "type": "string", "description": "Name or identifier of the allele-specific primer." }, "sequence": { "type": "string", "description": "Nucleotide sequence of the allele-specific primer (5' to 3').", "pattern": "^[ATCGatcg]+$" }, "length": { "type": "integer", "description": "Length of the allele-specific primer in bases.", "minimum": 1 }, "tm": { "type": "number", "description": "Melting temperature (Tm) of the allele-specific primer in °C.", "minimum": 0 }, "target_allele": { "type": "string", "description": "Allele targeted by the primer." }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "name", "sequence", "length", "target_allele", "reagent_details" ] } }, "competimer_primers": { "type": "array", "description": "Competitive primers used to modulate PCR efficiency.", "items": { "type": "object", "properties": { "name": { "type": "string", "description": "Name or identifier of the competimer primer." }, "sequence": { "type": "string", "description": "Nucleotide sequence of the competimer primer (5' to 3').", "pattern": "^[ATCGatcg]+$" }, "length": { "type": "integer", "description": "Length of the competimer primer in bases.", "minimum": 1 }, "modifications": { "type": "string", "description": "e.g., 3' amino modification to prevent extension." }, "percentage": { "type": "number", "description": "Percentage of competimer relative to total primer concentration.", "minimum": 0, "maximum": 100 }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "name", "sequence", "length", "percentage", "reagent_details" ] } } }, "required": [ "forward_primer", "reverse_primer" ] }, "allele_specific_oligonucleotides": { "type": "object", "description": "ASO probes for hybridization-based SNP detection.", "properties": { "probes": { "type": "array", "description": "List of ASO probes used in the experiment.", "items": { "type": "object", "properties": { "name": { "type": "string", "description": "Name or identifier of the ASO probe." }, "sequence": { "type": "string", "description": "Nucleotide sequence of the ASO probe (5' to 3').", "pattern": "^[ATCGatcg]+$" }, "length": { "type": "integer", "description": "Length of the ASO probe in bases.", "minimum": 1 }, "target_allele": { "type": "string", "description": "Allele targeted by the ASO probe." }, "labeling": { "type": "string", "description": "Labeling method for the ASO probe.", "enum": [ "radioactive", "biotin", "fluorescent", "none" ] }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "name", "sequence", "length", "target_allele", "reagent_details" ] } }, "hybridization_conditions": { "type": "object", "description": "Conditions under which ASO probes were hybridized to the target DNA.", "properties": { "temperature": { "type": "number", "description": "Hybridization temperature in °C.", "minimum": 0 }, "buffer": { "type": "string", "description": "Buffer used for hybridization." }, "wash_conditions": { "type": "object", "description": "Stringency wash conditions post-hybridization.", "properties": { "buffer": { "type": "string", "description": "Buffer used for washing." }, "temperature": { "type": "number", "description": "Wash temperature in °C.", "minimum": 0 }, "time": { "type": "number", "description": "Duration of the wash in minutes.", "minimum": 0 } }, "required": [ "buffer", "temperature" ] } }, "required": [ "temperature", "buffer" ] } } }, "fluorogenic_probes": { "type": "array", "description": "Fluorogenic probes for real-time PCR (TaqMan, molecular beacons, Scorpion primers, hybridization probes).", "items": { "type": "object", "properties": { "name": { "type": "string", "description": "Name or identifier of the fluorogenic probe." }, "sequence": { "type": "string", "description": "Nucleotide sequence of the fluorogenic probe (5' to 3').", "pattern": "^[ATCGatcg]+$" }, "length": { "type": "integer", "description": "Length of the fluorogenic probe in bases.", "minimum": 1 }, "probe_type": { "type": "string", "description": "Type of fluorogenic probe.", "enum": [ "TaqMan", "molecular beacon", "Scorpion primer", "hybridization probe", "other" ] }, "labeling": { "type": "object", "description": "Fluorescent dyes used for labeling the probe.", "properties": { "reporter_dye": { "type": "string", "description": "Reporter fluorescent dye.", "enum": [ "FAM", "VIC", "TET", "HEX", "CY3", "CY5", "ROX", "other" ] }, "quencher_dye": { "type": "string", "description": "Quencher fluorescent dye.", "enum": [ "TAMRA", "BHQ1", "BHQ2", "MGB", "NFQ", "none", "other" ] } }, "required": [ "reporter_dye", "quencher_dye" ] }, "target_description": { "type": "string", "description": "Description of the target sequence." }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "name", "sequence", "length", "labeling", "reagent_details" ] } }, "dna_polymerase": { "type": "object", "description": "Name of thermostable DNA polymerase enzyme used. Determines process efficiency and fidelity.", "properties": { "type": { "type": "string", "description": "Type of DNA polymerase.", "enum": [ "Taq", "Pfu", "Phusion", "Klenow fragment", "Tth", "reverse transcriptase", "other" ] }, "heat_stable": { "type": "boolean", "description": "Indicates if the polymerase is heat-stable or thermolabile.", "default": true }, "nuclease_activity": { "type": "string", "description": "Type of nuclease activity.", "enum": [ "5'→3' exonuclease", "3'→5' exonuclease", "none" ] }, "reverse_transcriptase_activity": { "type": "boolean", "description": "Indicates if the polymerase has reverse transcriptase activity." }, "inhibitor_tolerance": { "type": "string", "description": "Description of polymerase tolerance to inhibitors." }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "type", "reagent_details" ] }, "dntps": { "type": "object", "description": "Deoxynucleotide triphosphates (dNTPs) as building blocks for DNA synthesis.", "properties": { "ratio": { "type": "object", "description": "Molar ratio of individual dNTPs (default 1:1:1:1).", "properties": { "dATP": { "type": "number", "description": "Ratio of dATP.", "minimum": 0 }, "dTTP": { "type": "number", "description": "Ratio of dTTP.", "minimum": 0 }, "dCTP": { "type": "number", "description": "Ratio of dCTP.", "minimum": 0 }, "dGTP": { "type": "number", "description": "Ratio of dGTP.", "minimum": 0 }, "dUTP": { "type": "number", "description": "Ratio of dUTP, if used.", "minimum": 0 } } }, "labeling": { "type": "string", "description": "Type of labeling for dNTPs.", "enum": [ "radioactive", "fluorescent", "none" ] }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "reagent_details" ] }, "buffer_system": { "type": "object", "description": "The buffer system that maintains optimal pH and ionic conditions for enzyme activity, often including Mg²⁺ concentration.", "properties": { "type": { "type": "string", "description": "Type of buffer used." }, "ph": { "type": "number", "description": "pH of the buffer system, optimized for DNA polymerase activity.", "minimum": 0, "maximum": 14 }, "components": { "type": "object", "description": "Buffer additives and modifiers, with their concentrations, that influence reaction specificity, stringency, and enzyme performance.", "properties": { "mgcl2_concentration": { "type": "number", "description": "Concentration of MgCl₂ in mM.", "minimum": 0 }, "salt_concentration": { "type": "number", "description": "Concentration of additional salts in mM.", "minimum": 0 }, "dmso_concentration": { "type": "number", "description": "Concentration of dimethyl sulfoxide (DMSO) in % (v/v), if used.", "minimum": 0 }, "betaine_concentration": { "type": "number", "description": "Concentration of betaine in mM, if used.", "minimum": 0 }, "formamide_concentration": { "type": "number", "description": "Concentration of formamide in % (v/v), if used.", "minimum": 0 }, "dtt_concentration": { "type": "number", "description": "Concentration of dithiothreitol (DTT) in mM, if used.", "minimum": 0 }, "tween_20_concentration": { "type": "number", "description": "Concentration of Tween 20 in % (v/v), if used.", "minimum": 0 } } }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "type", "ph", "reagent_details" ] }, "internal_controls": { "type": "object", "description": "Internal controls for normalization or validation (housekeeping genes, competitive templates, spike-ins).", "properties": { "type": { "type": "string", "description": "Type of internal control.", "enum": [ "housekeeping gene", "competitive template", "spike-in", "none" ] }, "target_description": { "type": "string", "description": "Description of the internal control target." } } }, "carryover_prevention": { "type": "object", "description": "Methods for preventing carryover contamination (UNG/dUTP).", "properties": { "method": { "type": "string", "description": "Method used for carryover prevention.", "enum": [ "UNG treatment", "dUTP incorporation", "physical separation", "none" ] }, "ung_treatment": { "type": "object", "description": "Parameters for UNG treatment if used.", "properties": { "temperature": { "type": "number", "description": "Temperature for UNG incubation in °C.", "minimum": 0 }, "time": { "type": "number", "description": "Duration of UNG incubation in minutes.", "minimum": 0 }, "reagent_details": { "$ref": "#/$defs/reagent" } }, "required": [ "temperature", "time", "reagent_details" ] } } } }, "required": [ "template_dna", "primers", "dna_polymerase", "dntps", "buffer_system" ] }, "thermal_cycling_conditions": { "type": "object", "description": "Full thermal cycling protocol including initial denaturation, amplification cycles, final extension, and optional hold.", "properties": { "instrument": { "type": "string", "description": "Model of thermal cycler (e.g., ABI Prism 7700, GeneAmp PCR System 9600, LightCycler, QX200 Droplet Digital PCR System, CFX384)." }, "initial_denaturation": { "type": "object", "description": "Initial step to separate double-stranded DNA into single strands.", "properties": { "temperature": { "type": "number", "description": "Temperature for initial DNA strand separation in degrees Celsius (°C).", "minimum": 0 }, "time": { "type": "number", "description": "Duration of initial denaturation step in seconds.", "minimum": 0 } }, "required": [ "temperature", "time" ] }, "reverse_transcription_step": { "type": "object", "description": "Optional reverse transcription step for RT-qPCR.", "properties": { "temperature": { "type": "number", "description": "Temperature for reverse transcription in °C.", "minimum": 0 }, "time": { "type": "number", "description": "Duration of reverse transcription in minutes.", "minimum": 0 } } }, "cycles": { "type": "array", "description": "Repeated amplification cycle steps.", "items": { "type": "object", "properties": { "denaturation": { "type": "object", "description": "Step to separate double-stranded DNA into single strands.", "properties": { "temperature": { "type": "number", "description": "Temperature in °C.", "minimum": 0 }, "time": { "type": "number", "description": "Duration in seconds.", "minimum": 0 } }, "required": [ "temperature", "time" ] }, "annealing": { "type": "object", "description": "Step where primers bind to complementary template sequences.", "properties": { "temperature": { "type": "number", "description": "Temperature in °C.", "minimum": 0 }, "time": { "type": "number", "description": "Duration in seconds.", "minimum": 0 } }, "required": [ "temperature", "time" ] }, "extension": { "type": "object", "description": "Step where DNA polymerase synthesizes new strands. Time typically proportional to amplicon length.", "properties": { "temperature": { "type": "number", "description": "Temperature in °C.", "minimum": 0 }, "time": { "type": "number", "description": "Duration in seconds.", "minimum": 0 } }, "required": [ "temperature", "time" ] }, "fluorescence_acquisition": { "type": "object", "description": "Real-time fluorescence monitoring parameters.", "properties": { "temperature": { "type": "number", "description": "Temperature at which fluorescence is acquired in °C.", "minimum": 0 }, "time": { "type": "number", "description": "Duration of fluorescence acquisition in seconds.", "minimum": 0 }, "acquisition_mode": { "type": "string", "description": "Mode of fluorescence acquisition.", "enum": [ "single", "continuous", "none" ] } } } }, "required": [ "denaturation", "annealing", "extension" ] } }, "final_extension": { "type": "object", "description": "Final extension step to complete DNA synthesis.", "properties": { "temperature": { "type": "number", "description": "Temperature in °C.", "minimum": 0 }, "time": { "type": "number", "description": "Duration in seconds.", "minimum": 0 } }, "required": [ "temperature", "time" ] }, "number_of_cycles": { "type": "integer", "description": "Total amplification cycles (e.g., 25 for PCR-ASO, 40 for qPCR).", "minimum": 1 }, "hold": { "type": "object", "description": "Optional post-cycling hold step.", "properties": { "temperature": { "type": "number", "description": "Temperature in °C.", "minimum": 0 }, "time": { "type": "number", "description": "Duration in seconds.", "minimum": 0 } } }, "ramp_rate": { "type": "number", "description": "Rate of temperature change between steps in °C/second." }, "melting_curve_analysis": { "type": "object", "description": "Post-amplification melting curve parameters.", "properties": { "start_temperature": { "type": "number", "description": "Starting temperature for melting curve analysis in °C.", "minimum": 0 }, "end_temperature": { "type": "number", "description": "Ending temperature for melting curve analysis in °C.", "minimum": 0 }, "increment": { "type": "number", "description": "Temperature increment per step in °C.", "minimum": 0 }, "hold_time": { "type": "number", "description": "Hold time at each temperature step in seconds.", "minimum": 0 } }, "required": [ "start_temperature", "end_temperature", "increment" ] }, "ddpcr_specific": { "type": "object", "description": "Parameters specific to droplet digital PCR.", "properties": { "droplet_generation": { "type": "object", "description": "Parameters for droplet generation in ddPCR.", "properties": { "oil_type": { "type": "string", "description": "Type of oil used for droplet generation." }, "droplet_volume": { "type": "number", "description": "Volume of each droplet in nanoliters.", "minimum": 0 }, "number_of_droplets": { "type": "integer", "description": "Total number of droplets generated.", "minimum": 1 } }, "required": [ "oil_type", "droplet_volume", "number_of_droplets" ] }, "droplet_reading": { "type": "object", "description": "Parameters for reading droplets in ddPCR.", "properties": { "instrument": { "type": "string", "description": "Model or type of droplet reader." }, "threshold_setting": { "type": "string", "description": "Method for setting threshold for positive droplets.", "enum": [ "automatic", "manual", "algorithmic" ] }, "rain_droplet_handling": { "type": "string", "description": "Method for handling rain (intermediate-amplitude) droplets.", "enum": [ "exclude", "include", "classify-separately" ] }, "poisson_correction": { "type": "boolean", "description": "Indicates whether Poisson correction was applied to account for droplet partitioning variability.", "default": true }, "limit_of_blank": { "type": "number", "description": "Limit of blank (LOB) for the ddPCR assay." }, "limit_of_detection": { "type": "number", "description": "Limit of detection (LOD) for the ddPCR assay." } }, "required": [ "instrument" ] } } } }, "required": [ "initial_denaturation", "cycles", "number_of_cycles", "final_extension" ] }, "amplicon": { "type": "object", "description": "Properties of the amplified DNA fragment.", "properties": { "length": { "type": "integer", "description": "Length of the amplicon in base pairs (bp).", "minimum": 1 }, "sequence": { "type": "string", "description": "Reference sequence of the target region.", "pattern": "^[ATCGatcg]+$" }, "gc_content": { "type": "number", "description": "Percentage of guanine (G) and cytosine (C) bases.", "minimum": 0, "maximum": 100 }, "target_description": { "type": "string", "description": "Description of the target." }, "restriction_sites": { "type": "array", "description": "Restriction enzyme sites in the amplicon.", "items": { "type": "object", "properties": { "enzyme": { "type": "string", "description": "Name of the restriction enzyme." }, "sequence": { "type": "string", "description": "Recognition sequence of the enzyme.", "pattern": "^[ATCGatcg]+$" }, "position": { "type": "integer", "description": "Position of the site in the amplicon (1-based indexing).", "minimum": 1 }, "polymorphic": { "type": "boolean", "description": "Indicates if the site is polymorphic." } }, "required": [ "enzyme", "sequence" ] } } }, "required": [ "length" ] }, "detection_method": { "type": "object", "description": "Method for visualizing or quantifying PCR products.", "properties": { "method": { "type": "string", "description": "Detection technique.", "enum": [ "gel electrophoresis", "autoradiography", "dot blot", "qPCR fluorescence", "sequencing", "radioactive labeling", "oligomer restriction", "real-time PCR", "melting curve analysis", "ddPCR", "other" ] }, "details": { "type": "object", "description": "Method-specific configuration and parameters for the selected detection approach.", "properties": { "gel_electrophoresis": { "type": "object", "description": "Details for gel electrophoresis detection.", "properties": { "gel_type": { "type": "string", "description": "Type of gel.", "enum": [ "agarose", "polyacrylamide", "NuSieve agarose", "alkaline agarose", "other" ] }, "gel_concentration": { "type": "number", "description": "Gel concentration in %.", "minimum": 0 }, "dye": { "type": "string", "description": "Staining dye." }, "buffer": { "type": "string", "description": "Electrophoresis buffer." }, "voltage": { "type": "number", "description": "Voltage applied during electrophoresis in V/cm.", "minimum": 0 }, "run_time": { "type": "number", "description": "Duration of electrophoresis in minutes.", "minimum": 0 } } }, "quantitative_analysis": { "type": "object", "description": "Real-time PCR quantitative analysis parameters.", "properties": { "instrument": { "type": "string", "description": "Model or type of real-time PCR instrument." }, "baseline_correction": { "type": "object", "description": "Parameters for baseline fluorescence correction.", "properties": { "method": { "type": "string", "description": "Method used for baseline correction.", "enum": [ "automatic", "manual", "user-defined" ] }, "cycle_range": { "type": "string", "description": "Cycle range used for baseline calculation." } }, "required": [ "method" ] }, "threshold": { "type": "number", "description": "Threshold value for Ct calculation.", "minimum": 0 }, "threshold_method": { "type": "string", "description": "Method used to set the threshold.", "enum": [ "automatic", "manual", "dynamic" ] }, "curve_analysis_method": { "type": "string", "description": "Algorithm used for qPCR curve analysis.", "enum": [ "Standard-Cq", "LinRegPCR", "FPLM", "DART", "5PSM", "MAK2", "FPK-PCR", "LRE-Emax", "LRE-E100", "Cy0", "PCR-Miner" ] }, "efficiency_model": { "type": "string", "enum": [ "constant", "continuously decreasing" ], "description": "Assumption about PCR efficiency across cycles." }, "cq_value": { "type": "number", "description": "Quantification cycle (Cq/Ct): cycle at which fluorescence exceeds threshold.", "minimum": 0 }, "cy0_value": { "type": "number", "description": "Cy0: intersection of the abscissa and the tangent at the inflection point of the Richards curve." }, "f0_value": { "type": "number", "description": "Initial fluorescence (F0) associated with target quantity, calculated as Fq/E^Cq." }, "fq_threshold": { "type": "number", "description": "Fluorescence threshold used to determine Cq." }, "fb_baseline": { "type": "number", "description": "Baseline fluorescence independent of amplification." }, "arn": { "type": "number", "description": "Normalized reporter fluorescence (ARn = Rn+ − Rn−)." }, "data_analysis": { "type": "object", "description": "Parameters for data analysis in real-time PCR.", "properties": { "method": { "type": "string", "description": "Method used for data analysis.", "enum": [ "Cq", "ΔΔCq", "standard curve", "sigmoidal curve fitting", "efficiency-based", "other" ] }, "normalization": { "type": "string", "description": "Method for normalization." }, "efficiency_calculation": { "type": "string", "description": "Method for calculating amplification efficiency." }, "dynamic_range": { "type": "string", "description": "Dynamic range of the assay in orders of magnitude." }, "limit_of_detection": { "type": "number", "description": "Limit of detection (LOD) for the assay in copies or ng." }, "limit_of_blank": { "type": "number", "description": "Limit of blank (LOB) for the assay." } }, "required": [ "method" ] }, "performance_metrics": { "type": "object", "description": "Performance metrics for evaluating qPCR curve analysis methods.", "properties": { "bias": { "type": "number", "description": "Ratio of mean F0 for highest vs. lowest input (expected = input fold difference, e.g., 10,000 for 4 log10 dilution series)." }, "precision": { "type": "number", "description": "Within-triplicate variance of observed F0 values." }, "resolution": { "type": "number", "description": "Smallest detectable fold-difference at 5% significance level." }, "linearity": { "type": "number", "description": "Variance due to deviation from the regression line of log(input) vs. log(F0)." }, "amplification_efficiency": { "type": "number", "description": "Amplification efficiency per cycle as fold increase (1–2).", "minimum": 0, "maximum": 2 }, "dynamic_range": { "type": "string", "description": "Dynamic range of the assay in orders of magnitude." } } }, "ddpcr_analysis": { "type": "object", "description": "Analysis parameters specific to ddPCR, including thresholding and error correction.", "properties": { "absolute_quantification": { "type": "boolean", "description": "Indicates if absolute quantification was performed." }, "poisson_correction": { "type": "boolean", "description": "Indicates whether Poisson correction was applied to account for droplet partitioning variability." }, "droplet_classification": { "type": "object", "description": "Parameters for droplet classification.", "properties": { "positive_droplets": { "type": "integer", "description": "Number of positive droplets detected.", "minimum": 0 }, "negative_droplets": { "type": "integer", "description": "Number of negative droplets detected.", "minimum": 0 }, "total_droplets": { "type": "integer", "description": "Total number of droplets analyzed.", "minimum": 1 }, "threshold_method": { "type": "string", "description": "Method used to set the threshold for droplet classification." } }, "required": [ "positive_droplets", "negative_droplets", "total_droplets" ] }, "mutation_detection": { "type": "object", "description": "Parameters for mutation detection in ddPCR.", "properties": { "mutation_type": { "type": "string", "description": "Type of mutation detected." }, "allele_frequency": { "type": "number", "description": "Allele frequency of the detected mutation.", "minimum": 0, "maximum": 1 }, "limit_of_detection": { "type": "number", "description": "Limit of detection for the mutation assay." } } } } } } }, "dot_blot": { "type": "object", "description": "Details for dot blot detection using ASO probes.", "properties": { "membrane_type": { "type": "string", "description": "Type of membrane used for dot blot." }, "denaturation_method": { "type": "string", "description": "Method used to denature DNA for dot blot." }, "fixation_method": { "type": "string", "description": "Method used to fix DNA to the membrane." }, "probe_label": { "type": "string", "description": "Labeling method for the probe." } } }, "autoradiography": { "type": "object", "description": "Details for autoradiographic detection.", "properties": { "exposure_time": { "type": "number", "description": "Duration of autoradiographic exposure in hours.", "minimum": 0 }, "temperature": { "type": "number", "description": "Temperature during exposure in °C." }, "intensification_screen": { "type": "boolean", "description": "Whether an intensification screen was used." } } } } } }, "required": [ "method" ] }, "restriction_analysis": { "type": "object", "description": "Post-PCR restriction enzyme digestion for allele discrimination (e.g., PCR-OR).", "properties": { "enzymes": { "type": "array", "description": "Restriction enzymes used.", "items": { "type": "string" } }, "digestion_conditions": { "type": "object", "description": "Reaction conditions used for the restriction enzyme digestion.", "properties": { "temperature": { "type": "number", "description": "Temperature for digestion in °C.", "minimum": 0 }, "time": { "type": "number", "description": "Duration of digestion in minutes.", "minimum": 0 }, "buffer": { "type": "string", "description": "Buffer used for digestion." } }, "required": [ "temperature", "time" ] }, "sequential_digestion": { "type": "boolean", "description": "Indicates if enzymes were used sequentially." } } }, "efficiency_metrics": { "type": "object", "description": "Amplification efficiency and yield metrics.", "properties": { "amplification_efficiency": { "type": "number", "description": "Efficiency per cycle as fold increase (1–2).", "minimum": 0, "maximum": 2 }, "total_amplification": { "type": "number", "description": "Total fold amplification.", "minimum": 0 }, "cq_value": { "type": "number", "description": "Quantification cycle (Cq/Ct) at which sample fluorescence crosses the detection threshold." }, "dynamic_range": { "type": "string", "description": "Dynamic range of the assay in orders of magnitude." }, "calculation_method": { "type": "string", "description": "Method used to calculate efficiency." }, "efficiency_model": { "type": "string", "description": "Assumption about PCR efficiency: whether it is constant across cycles or decreases progressively. Influences the choice of curve analysis method.", "enum": [ "constant", "continuously decreasing" ] } } }, "reaction_volume": { "type": "number", "description": "Total PCR reaction volume in µL.", "minimum": 0 }, "experimental_parameters": { "type": "object", "description": "Additional experimental conditions and observations.", "properties": { "specificity": { "type": "string", "description": "Description of amplification specificity." }, "mutagenesis": { "type": "object", "description": "Details of intentional mutations introduced via primers.", "properties": { "type": { "type": "string", "description": "Type of mutagenesis." }, "sequence_added": { "type": "string", "description": "Sequence added via primer modifications (5' to 3').", "pattern": "^[ATCGatcg]+$" }, "position": { "type": "integer", "description": "Position of the mutation in the amplicon (1-based indexing).", "minimum": 1 } } }, "notes": { "type": "string", "description": "Additional observations." }, "automation": { "type": "boolean", "description": "Indicates if the PCR process was automated." }, "contamination_control": { "type": "string", "description": "Methods used to control contamination." }, "inhibition_control": { "type": "string", "description": "Methods used to control or assess inhibition." } } }, "controls": { "type": "object", "description": "Experimental controls.", "properties": { "negative_control": { "type": "boolean", "description": "Description of negative control sample used to monitor for false-positive reactions." }, "wild_type_control": { "type": "boolean", "description": "Description of wild-type control sample containing exclusively the wild-type sequence for comparison." }, "no_template_control": { "type": "boolean", "description": "Indicates if a no-template control was used." }, "no_reverse_transcriptase_control": { "type": "boolean", "description": "Indicates if a no-reverse transcriptase control was used for RT-qPCR experiments." }, "positive_control": { "type": "boolean", "description": "Positive control details." } } } }, "required": [ "experiment_metadata", "reaction_components", "thermal_cycling_conditions", "amplicon", "detection_method", "reaction_volume" ], "unevaluatedProperties": false }