User manual

Droplet Digital
™
PCR Applications Guide | 39
Copy Number Variation Analysis
CNV Calculations
CNV analysis by ddPCR involves quantification of target and reference loci through the use
of duplex target and reference assays. In QuantaSoft
™
software, copy number is determined
by calculating the ratio of the target molecule concentration to the reference molecule
concentration, times the number of copies of reference species in the genome (usually 2).
CNV = N
B
A = concentration of target species
B = concentration of reference species
N
B
= number of copies of reference loci in the genome (usually 2)
The error bars on a CN estimate in QuantaSoft software are the 95% confidence interval
of this measurement.
CNV Analysis in Homogeneous Samples
Analysis of CNV in homogeneous samples involves evaluation of CN state from a sample
source where every cell is presumed to be identical in CN state. Examples include
CYP2D6 genotyping in patient samples, trisomy 21 detection in a clonally derived Down
syndrome cell line, and counting transgene copies in a genetically modified organism (GMO)
plant stock. The challenge in homogeneous samples is largely a function of the level of
discrimination required at a high CN state to differentiate between adjacent CN states.
For some applications, for example, binning cancer patient samples by oncogene
amplification status, discrimination between high CN states (for instance, 50 vs. 51 copies)
may not be as critical as discriminating between CN states of 2 vs. 10 vs. 50.
CNV Analysis in Heterogeneous Samples
Some sample types are heterogeneous for CN state — meaning that for a given sample
of cells, some have altered CN status and some do not. Examples include a breast cancer
biopsy, where only a fraction of the cells are amplified for HER2, detection of fetal trisomy 21
from a maternal blood sample, or somatic mosaicism in normal tissue. In heterogeneous
samples, the ability to accurately quantify CN alterations is a function of both the percentage
of assayed cells with a CN alteration and the increase in CN state of the target gene in
those cells. It is more challenging to detect CNV for a target locus in a sample where 10%
of the cells have CN 3 instead of 2 than in a sample where 10% of the cells have a CN of
10 instead of 2. The problem becomes more difficult as the percentage of cells with the CN
alteration decreases. Detection of CN variant cells in a heterogeneous sample is a function
of both the magnitude of the CN alteration and the rarity of the altered cell (Table 4.1).
A
B