title: Targeted, haplotype-resolved resequencing of long segments of the human genome.
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.
authors.affiliation: University of Washington Genome Center, Department of Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Raymond
authors.forename: Sandhya
authors.initials: S
authors.lastname: Subramanian
authors.forename: Marcia
authors.initials: M
authors.lastname: Paddock
authors.forename: Ruolan
authors.initials: R
authors.lastname: Qiu
authors.forename: Chloe
authors.initials: C
authors.lastname: Deodato
authors.forename: Anthony
authors.initials: A
authors.lastname: Palmieri
authors.forename: Jean
authors.initials: J
authors.lastname: Chang
authors.forename: Tana
authors.initials: T
authors.lastname: Radke
authors.forename: Eric
authors.initials: E
authors.lastname: Haugen
authors.forename: Arnie
authors.initials: A
authors.lastname: Kas
authors.forename: David
authors.initials: D
authors.lastname: Waring
authors.forename: Donald
authors.initials: D
authors.lastname: Bovee
authors.forename: Robin
authors.initials: R
authors.lastname: Stacy
authors.forename: Rajinder
authors.initials: R
authors.lastname: Kaul
authors.forename: Maynard V
authors.initials: MV
authors.lastname: Olson
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D015894:Genome, Human / Q000235:genetics*; D005838:Genotype; D006239:Haplotypes / Q000235:genetics*; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*