Cycle threshold values were converted to relative copy numbers by using a standard curve of a sample with a known copy number.
Doubling of the copy number by using a tandem repeat of an antisense fragment did not further enhance the antisense effect, possibly indicating that a saturation point was already reached.
Further, retroviral transduction was followed for both cell lines for two reasons: first, this methodallows to statistically adjust the copy number by using a low, defined ratio of recombinant virus particles to the number of infected cells (multiplicity of infection).
Briefly, DNA samples were diluted from 10 to 10 viral genomic copies (vgc)/μL by using a quantified RFAV DNA template and were amplified by using AV3 primers with the detection threshold of ≈100 vgc in a volume of 15 μL.
In addition, genomic imbalances were identified using aCGH-Smooth which uses a genetic local search algorithm to identify breakpoints defining segmental DNA copy number changes by using a maximum likelihood estimation to optimise breakpoint location (Jong et al, 2004).
This function incorporates copy number call uncertainty by using a latent class model as described in [ 4].
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Absolute copy number quantification was performed by using a standard miR30a probe at different dilutions (Additional file 4: Figure S3A and Figure S3B).
The copy number differences were determined by using a standard ΔCt method that compares the mean Ct value of the target CNV fragments, determined from different input concentrations, compared to the mean Ct value of a known diploid reference.
To facilitate the identification of subclonal mutations in PD4120a, for which the genome was sequenced to an average 188-fold depth, we ran our substitution-calling algorithm by using a copy number of 10 throughout the tumor genome.
