The concentration of each plasmid was calculated based on absorbance at 260 nm, and a dilution series produced for calculation of copy number via qPCR.
Then, the concentration of P. putida KT2440 DNA was converted to a genome equivalent for calculation of copy numbers in the real-time PCR assays (Cottyn et al. 2011).
As an example of the type of genome-based information that can be obtained, we presented the successful calculation of copy number alterations and mutations from snap frozen as well as from FFPE breast tumor tissue.
Calculation of copy numbers (copy no).
Integrated densities used for the calculation of copy numbers were obtained using ImageJ software.
(i) Calculation of copy number profiles is mainly done as described in our previous publication (Boeva et al., 2010).
Calculation of copy number was based on the concentrations determined by a DU730 spectrophotometer (Beckman Coulter, Brea, CA, USA).
The presence of the negative control in each qPCR run is fundamental for the final calculation of copy number change of each marker.
Since none of the methods that we tested improved the yield, we used RNAs extracted with the hot-phenol method for the experiment but used the value obtained by the NaOH/PCA method for the calculation of copy number.
Here, we present the tool Control-FREEC that enables automatic calculation of copy number and allelic content profiles from NGS data, and consequently predicts regions of genomic alteration such as gains, losses and LOH.
Besides amplifications and deletions detected by both CGH and SNP arrays, SNP arrays can reveal mosaicism, extended regions of loss of heterozygosity, uniparental disomy (Conlin et al., 2010), provide more accurate calculation of copy numbers (Greenman et al., 2010) and determine parental origin of de novo CNVs (Conlin et al., 2010) in trios.
