A genome assembly-integrated dog 1 Mb BAC microarray: a cytogenetic resource for canine cancer studies and comparative genomic analysis.
Bottom Line: The emergence of high quality genome assemblies for several model organisms provides exciting opportunities to develop novel genome-integrated molecular cytogenetic resources that now permit a comparative approach to evaluating the relevance of tumor-associated chromosome aberrations, both within and between species.This panel of BAC clones also represents a powerful cytogenetic resource with numerous potential applications.We also show how individual clones selected from the BAC panel can be used as FISH probes in direct evaluation of tumor karyotypes, to verify and explore CNAs detected using aCGH analysis.
Affiliation: Department of Molecular Biomedical Sciences, College of Veterinary Medicine, Raleigh, NC 27606, USA.Show MeSH
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Mentions: The remaining 73 (40%) clones failed to hybridize to the expected location. These included three clones that failed to revive in culture, most likely due to a fault during library replication, or loss of the BAC vector resulting in loss of antibiotic resistance. Forty clones did not demonstrate any detectable hybridization signal after multiple attempts on metaphase chromosome preparations from a minimum of two unrelated dogs. A total of 27 clones mapped to a location inconsistent with their position in the genome assembly. Nineteen of these anomalies were resolved by selecting alternate clones from the assembly that share contiguous sequence with the original BAC, and thus were unlikely to reflect major inconsistencies between the cytogenetic map and the genome assembly. Finally, three clones (182G07, 307O16 and 376D10) mapped to the expected chromosome but at a location inconsistent with the genome assembly, and were further investigated. For clones 182G07 and 307O16 (from CFA1 and 6 respectively), the anomaly was resolved by selecting alternate clones from the genome assembly, which mapped to the expected location (see Fig. 2 for details). The observed correlation between genome sequence and FISH data for these alternate clones, positioned 0.3 Mb and 0.1 Mb respectively from the original selections, implies that the phase I findings represent minor discrepancies in the placement of individual clone sequences within the genome assembly for CFA1 and CFA6 rather than extensive discordance between the assembly and the cytogenetic map. A third clone, 376D10, originally placed at CFA2; 4.3 Mb according to BLAST of BAC end sequences, mapped significantly more distally than expected, and through co-hybridization with clones from this region, was resolved to approximately CFA2; 87 Mb. This finding was subsequently attributed to misplacement in the assembly of one end of the BAC clone sequence at CFA2; 4.3 Mb, most likely due to the presence of repetitive sequence, whilst the other end was correctly positioned at CFA2; 86.7 Mb. This region of CFA2, extending from 4 Mb to 11 Mb from the centromere, showed a unique profile of FISH results that reflect the existence of an extensive region of tandemly repeated DNA sequences, an example of which is given in Fig. 3. Details of clones with anomalous FISH data are provided in the supplementary online material associated with this report (http://www.cvm.ncsu.edu/mbs/breen_matthew.htm - table C).
Affiliation: Department of Molecular Biomedical Sciences, College of Veterinary Medicine, Raleigh, NC 27606, USA.