Abstract
When combined with haplotype fusion PCR (HF-PCR), ligation haplotyping is a robust, high-throughput method for empirical determination of haplotypes, which can be applied to assaying both sequence and structural variation over long distances. Unlike alternative approaches to haplotype determination, such as allele-specific PCR and long PCR, HF-PCR and ligation haplotyping do not suffer from mispriming or template-switching errors. In this method, HF-PCR is used to juxtapose DNA sequences from single-molecule templates, which contain single-nucleotide polymorphisms (SNPs) or paralogous sequence variants (PSVs) separated by several kilobases. HF-PCR uses an emulsion-based fusion PCR, which can be performed rapidly and in a 96-well format. Subsequently, a ligation-based assay is performed on the HF-PCR products to determine haplotypes. Products are resolved by capillary electrophoresis. Once optimized, the procedure can be performed quickly, taking a day and a half to generate phased haplotypes from genomic DNA.
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Acknowledgements
This work was funded by the Wellcome Trust (grant no. 077014/Z/05/Z). The authors thank Chris Tyler-Smith for his work on the primary ligation haplotyping paper and for his invaluable comments on this paper. We also thank Robert Graham for statistically derived IRF5 haplotype data and Oxford Journals for permission to reuse figures from the primary research paper.
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M.E.H. and D.J.T. designed this study, performed the analysis and wrote the manuscript. All experiments were performed by D.J.T.
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Turner, D., Hurles, M. High-throughput haplotype determination over long distances by haplotype fusion PCR and ligation haplotyping. Nat Protoc 4, 1771–1783 (2009). https://doi.org/10.1038/nprot.2009.184
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DOI: https://doi.org/10.1038/nprot.2009.184
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