Completely phased genome sequencing through chromosome sorting

Yang, Hong; Chen, Xi; Wong, Wing Hung

doi:10.1073/pnas.1016725108

Cited by 104 publications

(146 citation statements)

References 26 publications

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“…Low-pass sequencing of genomes from isolated sperm cells is a relatively straightforward and effective means to generate chromosome-length haplotypes without the need for specialized equipment to isolate individual chromosomes (Ma et al 2010;Fan et al 2011;Yang et al 2011). Using genotype data from 16 sperm cells, a simple one-versus-all approach was sufficient to identify recombination breakpoints that were subsequently validated using parental genotypes.…”

Section: Discussionmentioning

confidence: 99%

“…Sequencing of paired-end reads (Levy et al 2007;McKernan et al 2009) or sequencing of long DNA fragments (Kitzman et al 2011;Suk et al 2011;Peters et al 2012) have been used to link multiple variant loci into large haplotype blocks (N50 values of up to 1.0 Mb), although none of these blocks span entire chromosomes. Other approaches involve the physical separation of chromosomes and include the use of somatic cell hybrids (Douglas et al 2001), polony sequencing (Zhang et al 2006), chromosome microdissection (Ma et al 2010) or chromosome sorting by fluorescenceactivated cell sorting (FACS) or microfluidic manipulation (Fan et al 2011;Yang et al 2011). Each of these yields chromosomelength haplotypes, although none has yet been implemented to achieve dense maps that include the majority of known variants in a genome.…”

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confidence: 99%

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Sequencing of isolated sperm cells for direct haplotyping of a human genome

et al. 2013

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There is increasing evidence that the phenotypic effects of genomic sequence variants are best understood in terms of variant haplotypes rather than as isolated polymorphisms. Haplotype analysis is also critically important for uncovering population histories and for the study of evolutionary genetics. Although the sequencing of individual human genomes to reveal personal collections of sequence variants is now well established, there has been slower progress in the phasing of these variants into pairs of haplotypes along each pair of chromosomes. Here, we have developed a distinct approach to haplotyping that can yield chromosome-length haplotypes, including the vast majority of heterozygous single-nucleotide polymorphisms (SNPs) in an individual human genome. This approach exploits the haploid nature of sperm cells and employs a combination of genotyping and low-coverage sequencing on a short-read platform. In addition to generating chromosome-length haplotypes, the approach can directly identify recombination events (averaging 1

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Sequencing of isolated sperm cells for direct haplotyping of a human genome

et al. 2013

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“…However, haplotyping is not a trivial exercise, and previous attempts to produce whole-chromosome haplotypes were generally technically difficult and required special equipment: For example, single chromosomes were obtained by FACS (fluorescence-activated cell sorting) (Yang et al 2011), microdissection (Ma et al 2010), or microfluidic separation (Fan et al 2011). As an alternative, HaploSeq was introduced , which takes advantage of the fact that chromosomes do not intermingle in the nucleus but occupy separate territories.…”

Section: Modeling the 3d Genomementioning

confidence: 99%

The second decade of 3C technologies: detailed insights into nuclear organization

2016

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The relevance of three-dimensional (3D) genome organization for transcriptional regulation and thereby for cellular fate at large is now widely accepted. Our understanding of the fascinating architecture underlying this function is based on microscopy studies as well as the chromosome conformation capture (3C) methods, which entered the stage at the beginning of the millennium. The first decade of 3C methods rendered unprecedented insights into genome topology. Here, we provide an update of developments and discoveries made over the more recent years. As we discuss, established and newly developed experimental and computational methods enabled identification of novel, functionally important chromosome structures. Regulatory and architectural chromatin loops throughout the genome are being cataloged and compared between cell types, revealing tissue invariant and developmentally dynamic loops. Architectural proteins shaping the genome were disclosed, and their mode of action is being uncovered. We explain how more detailed insights into the 3D genome increase our understanding of transcriptional regulation in development and misregulation in disease. Finally, to help researchers in choosing the approach best tailored for their specific research question, we explain the differences and commonalities between the various 3C-derived methods.

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“…Several experimental phasing methods have been described. Examples include somatic cell hybrid construction to convert a diploid cell into haploid cell lines (2); large-insert cloning followed by screening of clone pools by either PCRbased genotyping (3) or sequencing (4)(5)(6) and isolation of individual chromosomes through chromosome microdissection of lysed metaphase cells on a glass slide (7)(8)(9); FACS-mediated single chromosome sorting (10); or the use of a custom microfluidic device (11) followed by amplification of the individual chromosomes and genotyping. The requirement of high-level expertise is a common denominator in these methods, which are not amenable to high-throughput studies or easily transferable to a clinical or diagnostic setting.…”

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confidence: 99%

Whole-genome haplotyping by dilution, amplification, and sequencing

Kaper

Swamy

Klotzle

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Standard whole-genome genotyping technologies are unable to determine haplotypes. Here we describe a method for rapid and cost-effective long-range haplotyping. Genomic DNA is diluted and distributed into multiple aliquots such that each aliquot receives a fraction of a haploid copy. The DNA template in each aliquot is amplified by multiple displacement amplification, converted into barcoded sequencing libraries using Nextera technology, and sequenced in multiplexed pools. To assess the performance of our method, we combined two male genomic DNA samples at equal ratios, resulting in a sample with diploid X chromosomes with known haplotypes. Pools of the multiplexed sequencing libraries were subjected to targeted pull-down of a 1-Mb contiguous region of the X-chromosome Duchenne muscular dystrophy gene. We were able to phase the Duchenne muscular dystrophy region into two contiguous haplotype blocks with a mean length of 494 kb. The haplotypes showed 99% agreement with the consensus base calls made by sequencing the individual DNAs. We subsequently used the strategy to haplotype two human genomes. Standard genomic sequencing to identify all heterozygous SNPs in the sample was combined with dilution-amplification-based sequencing data to resolve the phase of identified heterozygous SNPs. Using this procedure, we were able to phase >95% of the heterozygous SNPs from the diploid sequence data. The N50 for a Yoruba male DNA was 702 kb whereas the N50 for a European female DNA was 358 kb. Therefore, the strategy described here is suitable for haplotyping of a set of targeted regions as well as of the entire genome.genotype | next generation sequencing | phasing

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Completely phased genome sequencing through chromosome sorting

Cited by 104 publications

References 26 publications

Sequencing of isolated sperm cells for direct haplotyping of a human genome

Sequencing of isolated sperm cells for direct haplotyping of a human genome

The second decade of 3C technologies: detailed insights into nuclear organization

Whole-genome haplotyping by dilution, amplification, and sequencing

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