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A deep dive into every possible configuration parameter in PrimerLab. Use this guide to fine-tune your designs.

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File Structure

The configuration file is a YAML document divided into 5 main sections:
  1. input
  2. workflow
  3. parameters
  4. filtering
  5. output

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1. Input (input)

Defines the source of your DNA sequence. 
input:
  # Path to local FASTA file (Recommended)
  sequence_path: "./data/gene.fasta"
  
  # OR direct sequence string (For short tests)
  sequence: "ATGC..."
  
  # For batch mode, this is ignored and CLI input is used

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2. Workflow (workflow)

Selects the design engine. 
workflow: pcr   # Standard PCR
# OR
workflow: qpcr  # Quantitative PCR

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3. Parameters (parameters)

The core design constraints.

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tm (Melting Temperature)

Uses nearest-neighbor thermodynamics (SantaLucia 1998). 
tm:
  min: 57.0  # Minimum allowed Tm
  opt: 60.0  # Target Tm (Score penalty moves away from here)
  max: 63.0  # Maximum allowed Tm

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primer_size

Length of the primer in base pairs. 
primer_size:
  min: 18
  opt: 20
  max: 25

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product_size

Length of the resulting amplicon. 
product_size:
  min: 100
  max: 300
Tip: For qPCR, keep max below 150 bp for best efficiency.

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gc (GC Content)

Percentage of G and C bases. 
gc:
  min: 30.0
  max: 70.0
  clamp: 1   # Enforce at least 1 G/C at 3' end (0 to disable)

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4. qPCR Specific (probe)

Only used if workflow: qpcr. 
probe:
  tm:
    min: 68.0
    opt: 70.0  # Should be ~10°C higher than primer Tm
    max: 72.0
  size:
    min: 18
    max: 30
  exclude_5_g: true  # Avoid G at 5' end (quenches fluorophore)

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5. Filtering & Weights (filtering, scoring)

Advanced control over the ranking algorithm. 
filtering:
  max_poly_x: 5       # Reject runs of 5+ identical bases (e.g., AAAAA)
  max_self_dimer: -5.0 # Max delta G for self-dimer (kcal/mol)
  max_hairpin: -2.0    # Max delta G for hairpin

scoring:
  weights:
    tm: 1.0     # Importance of Tm accuracy
    gc: 0.5     # Importance of GC
    dimer: 2.0  # Importance of avoiding dimers (High!)
    size: 0.5   # Importance of exact product size