Fiber-FISH Analysis of Genomic Loci

A minimum of 20 informative fibers were scored for every single probe combination on an Olympus BX60 epifluorescence microscope equipped with Isis Imaging System (Metasystems). Fiber-FISH analyses were developed by applying the following assessment criteria adapted from Molina et al.50 (link): (i) at least three signals of different colour combinations must be observed in a consecutive and linear fashion, ii) two or more signals of the same colour are considered independent when they are separated by a distance twice the distance of every single bead-on-string, and iii) signals are considered informative regardless of the size (Fig. 1D). For each BAC and fosmid combination, we scored the frequency of the fosmid signal numbers that corresponded to the tandem sequence blocks.