Targeted, haplotype-resolved resequencing of long segments of the human genome

Genomics. 2005 Dec;86(6):759-66. doi: 10.1016/j.ygeno.2005.08.013. Epub 2005 Oct 24.

Abstract

Currently, challenges exist to acquire long-range (hundreds of kilobase pairs) phase-discriminated sequence across substantial numbers of individuals. We have developed a straightforward method for isolating and characterizing specific genomic regions in a haplospecific manner. Real-time PCR is carried out to STS content map and genotype pools of fosmid clones arrayed in 384-well microtiter plates. Single-nucleotide polymorphisms, microsatellite markers, and insertion-deletion polymorphisms are used to differentiate the target region into haplotype-specific tiling paths. DNA of clones from these tiling paths is retrieved from the library and either sequenced by standard shotgun methods or amplified in vitro and sequenced by a primer-based, directed method. This approach provides convenient access to complete, haplotype-resolved resequencing data from multiple individuals across tens to hundreds of thousands of basepairs. We illustrate its implementation with a detailed example of more than 400 kbp from the human CFTR region, across 15 individuals, and summarize our experience applying it to many other human loci.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cloning, Molecular / methods
  • Genome, Human / genetics*
  • Genotype
  • Haplotypes / genetics*
  • Humans
  • Microsatellite Repeats / genetics
  • Molecular Sequence Data
  • Polymorphism, Single Nucleotide / genetics
  • Sequence Analysis, DNA / methods*

Associated data

  • GENBANK/AY848828
  • GENBANK/AY848829
  • GENBANK/AY848830
  • GENBANK/AY848831
  • GENBANK/AY848832
  • GENBANK/AY849794