Assaying chromosomal inversions by single-molecular haplotyping

Inversions are an important form of structural variation, but are difficult to characterize as their breakpoints often fall within inverted repeats. We have developed a novel method, called ‘Haplotype Fusion’, in which an inversion breakpoint is genotyped by performing Fusion-PCR on single molecules of DNA. Fusing single copy sequences bracketing an inversion breakpoint generates orientation-specific PCR products, as exemplified by a genotyping assay for the int22 hemophilia A inversion on Xq28. This method is suitable for surveying inversion polymorphism at most inverted repeats in the human genome. Furthermore, we demonstrate that inversion events with breakpoints embedded within long (>100kb) inverted repeats can be genotyped by Haplotype Fusion PCR followed by bead-based single molecule haplotyping on repeat-specific markers bracketing the inversion breakpoint. We illustrate this method by genotyping a Yp paracentric inversion sponsored by >300kb long inverted repeats. The generality of our methods for genotyping chromosomal inversions should catalyse our understanding of the contribution of inversions to genomic variation, inherited diseases and cancer