Configuration Reference

YAML configuration file reference for PrimerLab

Complete reference for all PrimerLab configuration parameters.

---

Configuration File Structure

input:
  sequence: "ATCGATCG..."  # or sequence_path
  sequence_path: /path/to/sequence.fasta

parameters:
  tm:
    min: 57.0
    opt: 60.0
    max: 63.0
  
  primer_size:
    min: 18
    opt: 20
    max: 25
  
  product_size:
    min: 100
    max: 300
  
  gc:
    min: 40.0
    max: 60.0

output:
  format: json  # json, csv, text
  directory: ./output

---

Parameter Reference

Input Parameters

Parameter	Type	Description
sequence	string	Raw DNA sequence
sequence_path	path	Path to FASTA file

Tm (Melting Temperature)

Parameter	Default	Range	Description
tm.min	57.0	50-70	Minimum Tm
tm.opt	60.0	55-65	Optimal Tm
tm.max	63.0	58-72	Maximum Tm

Primer Size

Parameter	Default	Range	Description
primer_size.min	18	15-25	Minimum length
primer_size.opt	20	18-22	Optimal length
primer_size.max	25	20-30	Maximum length

GC Content

Parameter	Default	Description
gc.min	40.0	Minimum GC%
gc.opt	50.0	Optimal GC% (v0.8.0)
gc.max	60.0	Maximum GC%

Sequence Composition & Filtering (v0.8.0)

Parameter	Default	Description
max_poly_x	4	Maximum allowed mononucleotide repeat length
max_ns	0	Maximum allowed Ns in primer
max_tm_diff	5.0	Maximum Tm difference between forward and reverse primers
num_candidates	50	Number of primer pairs to generate
included_region	None	Dict with start and length to restrict design
force_left_start/end	None	Force forward primer coordinates
force_right_start/end	None	Force reverse primer coordinates
must_match_five_prime	None	5’ end constraint pattern
must_match_three_prime	None	3’ end constraint pattern

Product Size

Parameter	Default	Description
product_size.min	100	Minimum amplicon
product_size.max	300	Maximum amplicon

Recommendations by workflow:

Workflow	Min	Max	Notes
Standard PCR	200	1000	Flexible
qPCR	70	150	Short preferred
Long-range PCR	1000	10000	Specialized enzymes

---

qPCR-Specific Parameters

parameters:
  probe:
    tm:
      min: 65.0
      opt: 68.0
      max: 72.0
    size:
      min: 20
      max: 30

Parameter	Default	Description
probe.tm.opt	68.0	Probe Tm (5-10°C > primers)
probe.size.min	20	Minimum probe length
probe.size.max	30	Maximum probe length

---

Advanced Parameters

Thermodynamics and Salt Concentrations

PrimerLab natively uses Primer3’s ThermoAnalysis engine to calculate precise melting temperatures and thermodynamic penalties.

parameters:
  thermodynamics:
    salt_monovalent: 50.0      # mM (Na+, K+), default: 50.0
    salt_divalent: 1.5         # mM (Mg2+), default: 1.5
    dntp_conc: 0.6             # mM, default: 0.6
    dna_conc: 50.0             # nM, default: 50.0
    tm_method: santalucia      # santalucia or breslauer, default: santalucia
    salt_corrections: santalucia # santalucia, schildkraut, or owczarzy, default: santalucia

Parameter	Type	Default	Description
salt_monovalent	float	50.0	Concentration of monovalent cations (mM)
salt_divalent	float	1.5	Concentration of divalent cations like Mg²⁺ (mM)
dntp_conc	float	0.6	Concentration of dNTPs (mM)
dna_conc	float	50.0	Concentration of oligos (nM)
tm_method	enum	santalucia	Nearest-neighbor parameter set (santalucia or breslauer)
salt_corrections	enum	santalucia	Salt correction formula (santalucia, owczarzy, or schildkraut)

Thermodynamic Thresholds

parameters:
  thermo:
    hairpin_dg_max: -2.0    # kcal/mol
    homodimer_dg_max: -5.0  # kcal/mol
    heterodimer_dg_max: -5.0

Advanced Primer3 Parameters (v0.8.0)

parameters:
  qc_method: threshold # 'threshold' (using dG) or 'any' (using alignment scores)
  max_self_any_th: 47.0
  max_self_end_th: 47.0
  max_pair_compl_any_th: 47.0
  max_pair_compl_end_th: 47.0
  max_hairpin_th: 47.0
  
  weights:
    tm_gt: 1.0
    tm_lt: 1.0
    size_gt: 1.0
    size_lt: 1.0
    gc_percent_gt: 1.0
    gc_percent_lt: 1.0
    end_stability: 1.0

---

Output Configuration

output:
  format: json       # json, csv, text, html
  directory: ./output
  include_audit: true
  include_rationale: true

---

Example Configurations

Standard PCR

input:
  sequence_path: ./gene.fasta

parameters:
  tm:
    opt: 60.0
  product_size:
    min: 200
    max: 500

qPCR with TaqMan Probe

input:
  sequence_path: ./target.fasta

parameters:
  tm:
    opt: 60.0
  product_size:
    min: 70
    max: 150
  probe:
    tm:
      opt: 68.0

Long-Range PCR

input:
  sequence_path: ./region.fasta

parameters:
  tm:
    opt: 65.0
  product_size:
    min: 2000
    max: 10000
  primer_size:
    opt: 25

---

See Also

• Getting Started
• CLI Reference
• Examples