Direct determination of haplotypes from single DNA molecules

Abstract

Determining the long-range haplotypes in a diploid individual is a major technical challenge. Here we report a method of molecular haplotyping by directly imaging multiple polymorphic sites on individual human DNA molecules simultaneously. We demonstrate the utility of this technology by accurately determining the haplotypes consisting of up to 16 single-nucleotide polymorphisms in genomic regions up to 50 kilobases.The recent successful completion of the HapMap project 1 and the availability of low-cost, large-scale, high-throughput single-nucleotide polymorphism (SNP) genotyping platforms have paved the way for comprehensive genetic analysis of complex traits such as susceptibility to common diseases and drug responses through genome-wide association studies 2 . Haplotype analysis can enhance the statistical power in the mapping of human complex trait loci, with the potential of reducing the size of genome-wide association studies 3 . So far, the methods for determining haplotypes have considerable limitations that have prevented their use in large-scale and high-throughput genetic studies 4-7 .We had recently reported a DNA bar-coding method based on direct imaging of multiple polymorphic or sequence-motif sites on individual DNA molecules simultaneously [8][9][10] . Here we report a new molecular haplotyping approach, which improves the labeling efficiency and allows one to experimentally determine haplotypes of targeted genomic regions. Our method starts with long-range PCR amplification of target DNA segments containing the polymorphic sites followed by allele-specific labeling of polymorphic alleles with fluorescent dye molecules. The labeled double-stranded DNA (dsDNA) molecules are then stretched into linear form on a glass surface and imaged using multicolor total internal reflection fluorescence microscopy, a technique capable of localizing single fluorescent dye molecules with nanometer-scale accuracy 11 . By determining the order of the fluorescent labels on the backbone at all the polymorphic sites of fully labeled dsDNA fragments, the haplotype(s) of an individual can be inferred with great accuracy, in a manner similar to reading a bar code.Highly efficient allele-specific labeling of the markers on the long-range PCR products for single-molecule analysis is the most critical aspect of our haplotyping method. To increase the labeling efficiency, we generated single-stranded DNA (ssDNA) fragments from doublestranded long-range PCR products. We accomplished this by modifying one PCR primer with a 5′ Cy3 label and the other with 5′ phosphorylation in a PCR. When we incubated the resulting PCR product with lambda exonuclease, the DNA strand with the phosphorylated 5′ end was digested efficiently whereas the one with Cy3-modified 5′ end was protected from digestion and became the ssDNA template. We also used the end Cy3 label to orient the DNA molecules. Using this approach, we generated single ssDNA molecules up to 18 kilobases (kb) routinely. With ssDNA templates, the allele-specific extensi...

Completely phased genome sequencing through chromosome sorting

“…Ultimately, experimental approaches remain the most attractive solutions to the phasing problem. Current approaches include using emulsion PCR to condense polymorphic sites from a single template (7,8), genotyping from diluted aliquots of DNA fragments (9), allele-specific imaging of long-range PCR products (10), using long-range polony (11), genotyping from sperms (12), and isolating single chromosomes by interspecific cell fusion (13). Most of these methods were designed for haplotyping only a small number of markers.…”

Genome structural variation discovery and genotyping

“…DNA barcoding methodologies are a promising alternative that may one day allow high-throughput detection of balanced structural differences; these methods include scanning fluorescently nick-labelled DNA molecules in nano-channel flow cells 51 or nanoslits 52 and the use of SNP-specific labelling of stretched DNA for haplotype resolution 53 . Similarly, absolute copy-number estimations made by amplifying single molecules using emulsion picolitre droplet PCR 54 or single-molecule sequencing of human genomes 55 offer tremendous potential to understand structural changes at the cellular level.…”

DNA origami-based shape IDs for single-molecule nanomechanical genotyping

“…Among mainstream haplotyping approaches621222627, imaging-based three methods are highly attractive, as their direct reading feature complements NGS in rapid assembly of short-reads of several hundred base pairs and provides feasibility and effectiveness in targeted genomic region studies. Several elegant studies have demonstrated the effectiveness of fluorescence imaging in haplotyping2829. Nevertheless, the diffraction limit of 200–300 nm restricted the resolution to be of ∼1,000 bp.…”