Hardware and software requirements for quantitative analysis of comparative genomic hybridization

Abstract

Key terms: Background correction, CCD camera., filters, fluorochromes, image quality assessment. intensity normalization, pixel shift correction, relative copy number karyotypeComparative Genomic Hybridization (CGH) analysis (4) has rapidly established itself as a powerful method for analyzing whole genomic DNA (3, 5 , 6, 10, 14, 15), and many researchers are interested in acquiring their own CGH analysis capability. Although some chromosomal imbalances, e.g. high copy number amplifications, can be identified by conventional microscopy, comprehensive interpretation of CGH requires digital image analysis. However, at the time of writing there are no comrnercially available software packages which come close to the capabilities of the in-house systems used by the various research teams which have been at the forefront of CGH developments and application (9, 11, 13).At a recent workshop o n the topic of "CGH Imaging" (organised by the EC Concerted Action on Automation of Molecular Cytogenetic Analyses, and held at the MRC Human Genetics Unit, Edinburgh, 2-3 June 1994), three discussion sessions were devoted to specifying what could be considered to be an "adequate provision" of hardware and analysis software, and what guidelines should be used in the interpretation of CGH data. These are respectively the topics of the three sections of this report, which summarises the consensus that was reached at that workshop.We hope that these recommendations will prove to be useful for prospective users of CGH. This technical note is not intended to be an introduction to CGH, nor a sur. vey of the literature on the subject, nor is it concerned with preparative techniques (which are discussed in de.. tail in Refs. 7, 8). Readers are referred to original source articles for detailed information on biological preparation protocols, digital microscopy, and analysis software; and also for further discussion of the issues concerning interpretation of the results of CGH analyses, and limitations of the technique. HARDWARE REQUIREMENTS Microscope OpticsRecent microscope models from the major manufacturers, equipped for epi-fluorescence, are adequate. The objective should be "plan" and apochromatic with a high numerical aperture. Some comniercially available apochromatic objectives are not suitable for CGH analysis since they do not transmit UV excitation. Some recent "fluar" objectivcs have developed high levels of autofluorescence after short periods of use; users should check for lens autofluorescence regularly and replace the objective when necessary. Mercury arc lamp light sources are adequate if they are stable, and can be aligned to give uniform illumination without chromatic variation. Recent microscope models hare improved collector lenses that correct for chromatic aberrations of the illumination, but problems may be experienced with older models. FiltersFilter sets should be selected to minimize the crosstalk betwcen fluorochromes. Ideally, this would be achieved by using fluorochromes with completely separated excitation a...

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“…The central line in the pro®les represents the most frequently measured¯uorescence ratio for each metaphase spread, corresponding to a balanced state of chromosomal material. The right and left vertical lines de®ne threshold values for over-and underrepresentation of chromosomal material (du Manoir et al, 1995).…”

Significant Hybridization Differences in Comparative Genomic Hybridization due to Nucleotides Used for DNA Labelling and to DNA Chosen for Cohybridization

“…Whereas a normal karyotype was obtained with some of the reference DNAs, others, in particular reference DNA A, led to numerous deviations in the same samples. Inconsistent results due to different reference DNAs were also reported for chromosome 19 by duManoir et al [15], who found that the deviations disappeared after changing the reference DNA. The authors explained their observation by varying contents of short interspersed repetitive elements, leading to low suppression in the hybridization procedure.…”