Normalization of array-CGH data: influence of copy number imbalances

Here we demonstrate that copy number imbalances correlate with intensity in array-CGH data thereby causing problems for conventional normalization methods. We propose a strategy to circumvent these problems by taking copy number imbalances into account during normalization, and we test the proposed strategy using several data sets from the analysis of cancer genomes. In addition, we show how the strategy can be applied to conveniently define adaptive sample-specific boundaries between balanced copy number, losses, and gains to facilitate management of variation in tissue heterogeneity when calling copy number changes.We highlight the importance of considering copy number imbalances during normalization of array-CGH data, and show how failure to do so can deleteriously affect data and hamper interpretation.Microarray-based techniques for genome-wide investigation of copy number aberrations (CNAs) have recently gained much attention. Initially employing arrays developed for gene expression analysis [1], or low-density arrays produced from large-insert genomic clones such as bacterial artificial chromosomes (BACs) [2], the application has evolved rapidly. Currently, specialized high-density arrays with oligonucleotide probes or probes derived from BAC clones are predominately used. Two-channel array-based comparative genomic hybridization (aCGH) is a direct successor to conventional metaphase CGH [3]. In both cases, DNA from two samples are differentially labeled with fluorescent dyes and co-hybridized to immobilized genomic capture probes. By use of aCGH, DNA derived from tumor tissue can be compared with reference DNA, e.g., normal whole blood DNA, and genomic imbalances can effectively be investigated. The main advantage of aCGH over conventional CGH is the increased resolution achieved by microarrays with a large number of individual probes, routinely up to hundreds of thousands, covering the entire genome [4]. The power of aCGH has been demonstrated in tumor studi