Targeted, haplotype-resolved resequencing of long segments of the human genome.
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.
Raymond
Christopher K
CK
University of Washington Genome Center, Department of Medicine, University of Washington, Seattle, WA 98195, USA.
Subramanian
Sandhya
S
Paddock
Marcia
M
Qiu
Ruolan
R
Deodato
Chloe
C
Palmieri
Anthony
A
Chang
Jean
J
Radke
Tana
T
Haugen
Eric
E
Kas
Arnie
A
Waring
David
D
Bovee
Donald
D
Stacy
Robin
R
Kaul
Rajinder
R
Olson
Maynard V
MV
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*