Abstract
Inversions are an important form of structural variation, but they are difficult to characterize, as their breakpoints often fall within inverted repeats. We have developed a method called 'haplotype fusion' in which an inversion breakpoint is genotyped by performing fusion PCR on single molecules of human genomic DNA. Fusing single-copy sequences bracketing an inversion breakpoint generates orientation-specific PCR products, exemplified by a genotyping assay for the int22 hemophilia A inversion on Xq28. Furthermore, we demonstrated that inversion events with breakpoints embedded within long (>100 kb) 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 >300-kb-long inverted repeats. The generality of our methods to survey for, and genotype chromosomal inversions should help our understanding of the contribution of inversions to genomic variation, inherited diseases and cancer.
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Acknowledgements
This work was funded by the Wellcome Trust. We thank M. Jobling for the kind gift of DNA, M. Ross for insights into Xq28, J. McCafferty and W. Howat for help with imaging, J. Collins and A. Coffey for guidance on pulsed-field gel elecrophoresis, and P. Holliger and Z. Oliynyk for advice on preparation of emulsions.
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Supplementary information
Supplementary Fig. 1
Blinded genotyping of ten males of known int22h inversion status. (PDF 158 kb)
Supplementary Fig. 2
Pulsed Field Gel Electrophoresis of genomic DNA before and after emulsion preparation. (PDF 227 kb)
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Turner, D., Shendure, J., Porreca, G. et al. Assaying chromosomal inversions by single-molecule haplotyping. Nat Methods 3, 439–445 (2006). https://doi.org/10.1038/nmeth881
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DOI: https://doi.org/10.1038/nmeth881
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