A Cytogenetically Characterized, Genome-Anchored 10-Mb BAC Set and CGH Array for the Domestic Dog

Abstract

The generation of a 7.5x dog genome assembly provides exciting new opportunities to interpret tumor-associated chromosome aberrations at the biological level. We present a genomic microarray for array comparative genomic hybridization (aCGH) analysis in the dog, comprising 275 bacterial artificial chromosome (BAC) clones spaced at intervals of approximately 10 Mb. Each clone has been positioned accurately within the genome assembly and assigned to a unique chromosome location by fluorescence in situ hybridization (FISH) analysis, both individually and as chromosome-specific BAC pools. The microarray also contains clones representing the dog orthologues of 31 genes implicated in human cancers. FISH analysis of the 10-Mb BAC clone set indicated excellent coverage of each dog chromosome by the genome assembly. The order of clones was consistent with the assembly, but the cytogenetic intervals between clones were variable. We demonstrate the application of the BAC array for aCGH analysis to identify both whole and partial chromosome imbalances using a canine histiocytic sarcoma case. Using BAC clones selected from the array as probes, multicolor FISH analysis was used to further characterize these imbalances, revealing numerous structural chromosome rearrangements. We outline the value of a combined aCGH/FISH approach, together with a well-annotated dog genome assembly, in canine and comparative cancer studies.

Fluorescence in situ Hybridization Applied to Domestic Animal Cytogenetics

“…However, the genome sequencing programs carried out in our species of interest should allow the development of this type of material and open new opportunities of research for animal cytogeneticists as illustrated in the previous studies carried out in the dog [Thomas et al, 2007].…”

Canine cytogenetics – from band to basepair

“…Using web-based resources such as the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/ hgGateway) CHORI-82 clones may be selected, based either on their position within the genome or their specific gene/sequence content, and used as SLPs in FISH analysis. Using this process chromosome tiling sets of arrays have been generated with clone spacing at 10 Mb resolution (Thomas et al, 2007) and 1 Mb resolution (Thomas et al, submitted).…”

“…With this in mind, genome integrated canine BAC arrays have been developed with the panels of genome-anchored BACs described above. The first is a genome-wide BAC array comprising 275 clones spaced at intervals of approximately 10 Mb and also contains clones representing the dog orthologues of 31 genes implicated in human cancers (Thomas et al, 2007). This array has been shown to be of use for first pass assessment of gross genome-wide DNA copy number variation in canine tumors and provides a similar resolution to that produced by metaphase based CGH analysis, albeit without the need to be able to identify the dog chromosomes.…”

“…To overcome this, panels of cytogenetically verified chromosome-specific single locus probes have been generated as described above (Thomas et al, 2003c(Thomas et al, , 2007Courtay-Cahen et al, 2007). Since these SLP panels are generated from commercially available canine BAC libraries, and the identity of each clone used is publicly available, it is a simple process for any cytogenetics laboratory to purchase these clones and generate their own SLP panels.…”

Characterization of canine osteosarcoma by array comparative genomic hybridization and RT‐qPCR: Signatures of genomic imbalance in canine osteosarcoma parallel the human counterpart

“…Until recently, very few studies in canine OS have profiled genomic instability, largely because of the paucity of resources for canine genomic studies. The release of the annotated canine genome assembly facilitated the development of genome integrated molecular cytogenetic reagents for the dog (Thomas et al, , 2007(Thomas et al, , 2008, allowing more comprehensive cytogenetic studies to be performed. In an earlier study we used lowresolution (10-20 Mb) aCGH analysis of 38 cases of canine OS to identify a high degree of gross genomic instability, with extensive cytogenetic disorganization consistent with those observed in human OS (Thomas et al, 2009b(Thomas et al, , 2011.…”