User manual

40 | Droplet Digital
™
PCR Applications Guide
Table 4.1. Detection of CN variant cells in a heterogeneous sample.
Sensitivity Required for CN Determination in a Heterogeneous Sample
Diploid Copy Number
Cells with CN Alteration, % Wild Type Amplified Discrimination Needed, %
2 3 5
10 2 10 40
2 50 240
2 3 0.5
1 2 10 4
2 50 24
2 3 0.05
0.1 2 10 0.4
2 50 2
2 3 0.005
0.01 2 10 0.040
2 50 0.240
Planning CNV Experiments
Assay Design
For CNV ddPCR, target and reference assays are designed with different probe
fluorophores, generally FAM-target and HEX-reference (or VIC-reference).
When possible, target and reference assays should have amplicons of similar length.
This is particularly important for fragmented samples, like material extracted from formalin-
fixed, paraffin-embedded (FFPE) tissue. Because CNV calculates the ratio of target
concentration to reference concentration, if either target or reference amplicon site in the
genome is under-represented due to fragmentation, the ratio call will be off-integer.
Check for secondary (nonspecific) products using the UCSC In-Silico PCR site
(http://genome.ucsc.edu/cgi-bin/hgPcr?command=start). This is particularly important
for CN-variable targets or members of gene families because of the presence of highly
similar pseudogenes or homologs.
An ideal reference assay yields expected concentrations reproducibly and robustly across
standard reaction parameters, duplex partners, and sample sources. A reference assay
should be CN invariant. A good reference assay performs well across a wide range of
annealing/extension temperatures.
Examples of human genome reference loci:
■
RPP30 (NCBI gene 10556)
■
Ultraconserved sequences (PMID: 15131266)
Copy Number Variation Analysis