Validation of array CGH-detected deletions. (A) Validation of a deletion by comparative FISH. FISH images are shown for orangutan variant array BAC RP11-171I8. To control for variability in FISH conditions, human and orangutan cells were mixed on a slide prior to fixation. Hybridization with BAC RP11-171I8 (red) and an unrelated control probe, RP11-233C13 (green) is shown for human (HSA) and orangutan (PPY). The complete absence of signal was observed for RP11-171I8 against the orangutan metaphase chromosomes, but not RP11-233C13. A genomic deletion at least the size of the BAC (175 kb by PFGE; see text) was confirmed by STS content mapping and Southern Analysis (data not shown). (B) Deletion validation by primate BAC analysis and Southern blot. Human BAC clone RP11-75H24 showed a reduced relative fluorescent signal intensity ratio of chimpanzee compared with human (Table 1). Four chimpanzee BAC clones from libraries RP-43 and CHORI-251 orthologous to this site were obtained, end-sequenced, and restriction mapped. A ∼38-kb discrepancy in size was determined by comparing BAC PFGE size and BAC end sequence placement of the chimpanzee loci against both public and private assembly versions of the human genome. Analysis of the human sequence identified ∼40 kb-tandem duplications (black boxes labeled Proximal and Distal), both containing exons 1–3 of gene BC005998. Using a probe spanning exon 1 of BC005998, Southern analysis of HaeII andSpeI-digested human (RP11-75H24) and chimpanzee (RP43-35C3) BAC DNA show a loss of a duplication within the chimpanzee genome (HaeII and SpeI restriction sites are indicated by the H and S, respectively).