Bacterial artificial chromosome-emulation oligonucleotide arrays for targeted clinical array-comparative genomic hybridization analyses

Abstract

Purpose: The goal of this work was to test the ability of oligonucleotide-based arrays to reproduce the results of focused bacterial artificial chromosome (BAC)-based arrays used clinically in comparative genomic hybridization experiments to detect constitutional copy number changes in genomic DNA. Methods: Custom oligonucleotide (oligo) arrays were designed using the Agilent Technologies platform to give high-resolution coverage of regions within the genome sequence coordinates of BAC/P1 artificial chromosome (PAC) clones that had already been validated for use in previous versions of clone arrays used in clinical practice. Standard array-comparative genomic hybridization experiments, including a simultaneous blind analysis of a set of clinical samples, were conducted on both array platforms to identify copy number differences between patient samples and normal reference controls.Results: Initial experiments successfully demonstrated the capacity of oligo arrays to emulate BAC data without the need for dye-reversal comparisons. Empirical data and computational analyses of oligo response and distribution from a pilot array were used to design an optimized array of 44,000 oligos (44K). This custom 44K oligo array consists of probes localized to the genomic positions of Ͼ1400 fluorescence in situ hybridization-verified BAC/PAC clones covering more than 140 regions implicated in genetic diseases, as well as all clinically relevant subtelomeric and pericentromeric regions. Conclusions: Our data demonstrate that oligo-based arrays offer a valid alternative for focused BAC arrays. Furthermore, they have significant advantages, including better design flexibility, avoidance of repetitive sequences, manufacturing processes amenable to good manufacturing practice standards in the future, increased robustness because of an enhanced dynamic range (signal to background), and increased resolution that allows for detection of smaller regions of change. Genet Med 2008:10(4):278 -289. Key Words: array-CGH, oligonucleotide, focused microarray, CNV, chromosome abnormalities, mosaicismThe advent of array-based copy number analysis using comparative genomic hybridization (CGH) or non-CGH methods, including analysis of single nucleotide polymorphisms, has been a breakthrough in the detection of chromosomal copy number changes in the clinical setting. 1 This approach has been shown to be superior to both classical cytogenetic banding methods and fluorescence in situ hybridization (FISH)-based methods because of the greatly improved resolution and highly multiplexed nature of the method. [2][3][4] It is clear that this "molecular cytogenetic" methodology will continue to expand the capabilities for correlations between chromosomal aberrations and clinical phenotypes. This will be invaluable, not only for the diagnostic potential, but also for eventual discovery of the true genotypic basis for specific syndromic features at the molecular level.Until recently, most clinical applications of array-CGH, other than some cancer studies, have...

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