How do you calculate copy numbers?

Calculating Copy Numbers in Molecular Diagnostics

To calculate copy numbers in molecular diagnostics, use the formula: (sum of target gene copies)/(sum of reference gene copies) × conversion factor × 100, which provides the most accurate quantification for clinical decision-making. 1

Basic Principles of Copy Number Calculation

Copy number calculation is essential for accurate molecular monitoring, particularly in conditions like chronic myeloid leukemia (CML) where treatment decisions depend on precise quantification of disease burden.

Standard Formula for PCR-Based Copy Number Calculation:

• Formula: (sum of target gene copies)/(sum of reference gene copies) × CF × 100
• Where: CF = laboratory-specific conversion factor 1

This approach accounts for all replicates and standardizes results across different laboratories.

Step-by-Step Copy Number Calculation Process

1. Perform replicate PCR reactions (2-3 replicates recommended for routine analysis) 1

   • Ensure equal number of replicates for both target and reference genes
   • If different numbers of replicates are used, adjust calculations accordingly

2. Sum the copy numbers from all replicates for both target and reference genes

   • For detected target gene: Use actual copy number (minimum 3 copies per positive replicate)
   • For undetected target gene: Use 0 in the numerator 1

3. Apply the conversion factor specific to your laboratory

   • Example: If your lab's MMR equivalent is 0.08%, CF = 0.1/0.08 = 1.25 1

4. Calculate final percentage: (sum target)/(sum reference) × CF × 100

5. Quality control checks:

   • Ensure reference gene copies are sufficient (>10,000 ABL1 or >24,000 GUSB per replicate)
   • Replicates with insufficient reference gene copies should be considered inevaluable 1

Examples of Copy Number Calculations

Example 1: Both replicates positive

• BCR-ABL1 replicate 1: 7 copies
• BCR-ABL1 replicate 2: 3 copies
• ABL1 replicate 1: 24,000 copies
• ABL1 replicate 2: 28,000 copies
• Lab CF = 0.8
• Result = (10/52,000) × 0.8 × 100 = 0.015% 1

Example 2: One replicate positive, one negative

• BCR-ABL1 replicate 1: undetectable
• BCR-ABL1 replicate 2: 3 copies
• GUSB replicate 1: 43,000 copies
• GUSB replicate 2: 49,000 copies
• Lab CF = 1.8
• Result = (3/92,000) × 1.8 × 100 = 0.0059% 1

Special Considerations and Pitfalls

Reaction Volume Considerations

• For very small reaction volumes (e.g., 33 nl in OpenArray technology), calculate actual copy numbers in the reaction volume:
  • Copy number in reaction = (Starting concentration in copies/ml) × (reaction volume in ml) 2

Quality Control Requirements

• Minimum reference gene threshold: >10,000 ABL1 or >24,000 GUSB copies per replicate
• Positive replicate minimum: Count at least 3 copies for any positive replicate, even if fewer are detected 1
• Standard curve requirements: Efficiency between 90-110%, R² value >0.99 2

Common Pitfalls to Avoid

1. Insufficient reference gene copies leading to unreliable results
2. Not accounting for different numbers of replicates between target and reference genes
3. Ignoring reaction volume differences when comparing across platforms 2
4. Not applying appropriate conversion factors when standardizing between laboratories 1

Alternative Methods for Specific Applications

For other applications like transgenic research or CNV detection:

• Comparative CT method (2^-ΔΔCT): Useful for distinguishing twofold differences in gene copy numbers 3
• Pyrosequencing with paralogous sequences: Effective for highly similar genes 4
• High-depth sequencing (>60x): Recommended for cancer subclonal reconstruction 1

By following these guidelines, you can accurately calculate copy numbers for clinical decision-making in molecular diagnostics.