An overview of the Optical Mapping system. A: Single cells, obtained from a slice of the tumor biopsy, are purified by a percoll density gradient, then mixed with agarose and allowed to solidify in a mold, forming rectangular inserts. Prior to mapping, cells are lysed within the insert, the DNA electrophoretically extracted, elongated and immobilized on an Optical Mapping surface by means of capillary flow through a microchannel device. The lower half of panel A is a representative image of properly elongated DNA (long, white horizontal lines) after surface digestion, stained with YOYO-1. Microchannels are 100 μm wide as indicated by the scale bar (grey bar). B: Enlarged image of surface-bound genomic DNA, digested with SwaI, showing discrete restriction fragments separated by gaps. C: Automated machine vision detects DNA molecules (pseudocolored green), and calculates the mass of each fragment (white numbers), creating ordered restriction maps (Rmaps) from single DNA molecules (yellow bars). D and E: Strategy for constructing a genome-wide optical map starting from single molecule Rmaps. Rmaps are first clustered on a restriction map generated in-silico from the reference sequence of the human genome by pairwise alignment. Then consensus optical map contigs are constructed by de novo assembly of the Rmaps from a given window. Finally, the consensus map contigs are aligned back to the reference map, and differences are identified.