A novel SNP analysis method to detect copy number alterations with an unbiased reference signal directly from tumor samples

To address these limitations, we designed a novel "Virtual Normal" algorithm (VN), which allowed for construction of an unbiased reference signal directly from test samples within an experiment using any publicly available normal reference set as a baseline thus eliminating the need for an in-lab normal reference set.The algorithm was tested using an optimal, paired tumor/normal data set as well as previously uncharacterized pediatric malignant gliomas for which a normal reference set was not available. Using Affymetrix 250K Sty microarrays, we demonstrated improved signal-to-noise ratio and detected significant copy number alterations using the VN algorithm that were validated by independent PCR analysis of the target CNA regions.We developed and validated an algorithm to provide a virtual normal reference signal directly from tumor samples and minimize noise in the derivation of the raw CN signal. The algorithm reduces the variability of assays performed across different reagent and array batches, methods of sample preservation, multiple personnel, and among different laboratories. This approach may be valuable when matched normal samples are unavailable or the paired normal specimens have been subjected to variations in methods of preservation.DNA copy number alterations (CNA) including sequence amplifications and deletions can cause oncogene activation or reduced tumor suppressor gene function associated with the emergence of cancer [1,2]. Recent advances in microarray technology including high density SNP (single nucleotide polymorphism) profiling have provided a novel approach to evaluate CNA across the genome of patient tissue specimens as a potential diagnostic tool for tumor classification. However, detection of a copy number amplification or deletion is critically dependent on: 1) interrogation of SNPs and/or nonpolymorphic markers at a frequency sufficient to identify changes in specific genes, and 2) accurate detection of gene copy number in tumor tissue