Amplicon Analysis

Feature documentation: Amplicon Analysis

Analysis tools for evaluating amplicon quality after primer design.

Overview

The amplicon analysis module provides comprehensive quality assessment for PCR products including:

• Secondary Structure: Predicts potential problematic structures using ViennaRNA
• GC Profile: Analyzes GC content distribution across the amplicon
• GC Clamp: Evaluates G/C content at amplicon ends
• Melting Temperature: Predicts amplicon Tm using nearest-neighbor thermodynamics
• Quality Score: Combined 0-100 score with A-F grading

CLI Usage

Integrated with Primer Design

# Analyze amplicons after PCR primer design
primerlab run pcr --config design.yaml --amplicon-analysis

Output

When --amplicon-analysis is enabled, the output includes:

• Amplicon quality scores in the JSON output
• Quality warnings in the report

API Usage

from primerlab.api.public import analyze_amplicon

# Analyze an amplicon sequence
sequence = "ATGCGATCGATCGATCGATCGATCGATCGATCGATCG..."

result = analyze_amplicon(sequence)

print(f"Quality Score: {result['quality_score']}/100 ({result['grade']})")
print(f"Amplicon Tm: {result['amplicon_tm']['tm']}°C")
print(f"Warnings: {result['warnings']}")

Configuration

Default configuration file: config/amplicon_default.yaml

amplicon_analysis:
  secondary_structure:
    enabled: true
    dg_warning_threshold: -3.0  # kcal/mol
    
  gc_profile:
    enabled: true
    window_size: 50
    ideal_min: 40.0
    ideal_max: 60.0
    
  gc_clamp:
    enabled: true
    region_size: 5
    min_gc: 1
    max_gc: 3
    
  quality_score:
    enabled: true
    weights:
      structure: 0.30
      gc_uniformity: 0.25
      gc_clamp: 0.20
      length: 0.15
      tm_sharpness: 0.10

Quality Scoring

Grade	Score Range	Interpretation
A	85-100	Excellent amplicon quality
B	70-84	Good quality, minor issues
C	55-69	Acceptable, some concerns
D	40-54	Problematic, review recommended
F	0-39	Poor quality, redesign suggested

Warnings

The analyzer generates warnings for:

• Strong secondary structures (ΔG < -8 kcal/mol)
• Non-uniform GC content (>15% spread)
• Weak or strong GC clamps
• Amplicons outside optimal length range (100-500bp)