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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.

High-throughput haplotype determination over long distances by haplotype fusion PCR and ligation haplotyping