Figure 3

Comparison of methods used to detect novel copy number variations. Three methods employ comparative genomic hybridisation. In these methods, test (green bar) and reference (red bar) DNA are hybridised against: (A) a BAC array; [13] (B) a targeted BAC array; [15] or (C) a representational oligonucleotide array [31]. Probes of the array are drawn above the DNA region to which they hybridise. Darkened boxes within the test or reference sequence bars correspond to duplications only found in one of the two sequences. The colour of the probe indicates the relative hybridisation of the array to the assayed DNA, with yellow representing equal copy number, green representing a duplication of the test region and red representing a duplication of the reference region. (D) The single nucleotide polymorphism (SNP) gene chip can be used as a quantitative array [32] that assays only a test (green) DNA sequence. Quantification of copy number is obtained by comparing the intensity levels of 20 pairs of oligonucleotide probes per SNP (red lines) to data available for a reference set of control individuals. Both (C) and (D) use genomic representations created using restriction enzyme fragmentation followed by adaptor-mediated polymerase chain reaction (PCR). This PCR is optimised to amplify genomic DNA between 200 and 1,200 base pairs (bp). Thus, the regions illustrated are much smaller than in the other methods, demonstrated by their smaller scale. (E) and (F) use in silico genome comparisons. In (E), intra- or inter-assembly comparisons not only detect copy number changes (insertion/deletions; segments 2 and 3), but can also identify inversions (segments 7-9), sequence variations (segments 11a versus 11b) and rearrangements (segments 13 and 14). Segments 18-20 show how whole-genome shotgun (WGS) read depth can be used to detect duplications. A significant increase in the depth of WGS reads per 5 kilobases (kb) is often an indication of duplication (dark green box) in the genome [33]. In (F), the comparison is between the NCBI assembly (green) and a fosmid paired-end sequence (red) derived genome [16]. Fosmid paired-ends are shown above their corresponding sequence in the public assembly. The average size of the fosmid insert is ~40 kb. Significant deviations (< 32 kb or > 48 kb) from this mean could indicate a copy number variant and are highlighted. Abbreviations: BAC, bacterial artificial chromosome; Mb, megabase(s).