High-throughput haplotype determination over long distances by haplotype fusion PCR and ligation haplotyping.
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.
Turner
Daniel J
DJ
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. djt@sanger.ac.uk
Hurles
Matthew E
ME
D004247:DNA / Q000737:chemistry; D005796:Genes; D006239:Haplotypes; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
D004247:DNA