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44 | Droplet Digital
™
PCR Applications Guide
Copy Number Variation Analysis
Table 4.5. Sample loading considerations for higher-order CN targets (10–50 copies/diploid genome).
Reference Loading
for CN 2, CPD Reference, copies/µl Target, copies/µl
CN 2 CN 10 CN 20 CN 40
1 1,000 5,000 10,000 20,000
0.2 200 1,000 2,000 4,000
Target Loading for CN ≥10
CPD, copies per droplet; CN, copy number.
DNA Loading for Lower-Order CN Analysis (diploid CN <10)
For most routine CNV ddPCR applications, where a diploid target CN is expected to be
10 or less, approximately 0.2–1.0 reference gene copies per droplet (CPD) of DNA sample
should be loaded per well. This corresponds to 10–66 ng of human genomic DNA per well.
DNA Loading for Higher-Order CN Analysis (diploid CN >10)
When target CN is expected to exceed 10 copies/diploid genome, <15 ng of human
genomic DNA should be loaded per well. This ensures both target and reference copies fall
within the dynamic range of the instrument (Table 4.5). For example, if a reference locus is
loaded at 1 CPD (1,000 copies/µl), a target present 20 times more frequently per genome
is effectively loaded at 20 CPD (20,000 copies/µl), which is beyond the range of accurate
quantification of the instrument. If a reference locus is loaded at 0.2 CPD (200 copies/µl),
a target present 20 times more frequently is loaded at 4 CPD (4,000 copies/µl), which is
within the dynamic range of the instrument. For diploid CN >50, strategies where multiple
wells are used to estimate target and reference concentrations can be used.