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Haplotype-resolved genome sequencing: experimental methods and applications

Nature Reviews Genetics volume 16, pages 344–358 (2015)Cite this article

Subjects

Key Points

  • Haplotypes link together (that is, 'phase') groups of genetic variants that co-occur on single chromosomes. Although haplotypes have an important role in clinical genetics and association studies, they are not typically obtained by contemporary genotyping or sequencing technologies and must be determined separately.

  • Inferential methods for haplotype determination perform fairly poorly for the rare and private variants implicated in many genetic diseases. To phase this class of variants accurately and comprehensively, direct experimental methods are needed.

  • Dense haplotyping methods comprehensively phase variants into haplotype blocks at the scale of a single gene or a small number of genes and corresponding regulatory regions. Contiguity is defined within each block but not between adjacent or distant haplotype blocks.

  • Sparse haplotyping methods phase a more modest number of distant variants distributed along an entire chromosome or a chromosome arm. Resulting haplotypes are not comprehensive but have long-range contiguity that is currently unattainable using dense methods.

  • Reference panels of previously ascertained haplotypes can be used to correct errors in, or increase the density or contiguity of, directly obtained haplotypes. Such hybrid approaches yield improved haplotypes at low costs.

  • Although contiguity metrics are typically used to compare haplotype assemblies, comprehensive comparisons should also include measures of the accuracy, density and allele frequency spectrum of the phased variants.

Abstract

Human genomes are diploid and, for their complete description and interpretation, it is necessary not only to discover the variation they contain but also to arrange it onto chromosomal haplotypes. Although whole-genome sequencing is becoming increasingly routine, nearly all such individual genomes are mostly unresolved with respect to haplotype, particularly for rare alleles, which remain poorly resolved by inferential methods. Here, we review emerging technologies for experimentally resolving (that is, 'phasing') haplotypes across individual whole-genome sequences. We also discuss computational methods relevant to their implementation, metrics for assessing their accuracy and completeness, and the relevance of haplotype information to applications of genome sequencing in research and clinical medicine.

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Figure 1: Schematic of dense haplotyping methods.
Figure 2: Schematic of sparse haplotyping methods.
Figure 3: Combining direct and computational methods.
Figure 4: Haplotype contiguity and accuracy.

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Acknowledgements

The authors thank B. Browning, B. Vernot, A. Gordon and members of the Shendure Lab for discussions.

Author information

Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, 98195, Washington, USA

    Matthew W. Snyder & Jay Shendure

  2. Department of Molecular and Medical Genetics, Oregon Health & Sciences University, Portland, 97239, Oregon, USA

    Andrew Adey

  3. Department of Human Genetics, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Jacob O. Kitzman

  4. Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Jacob O. Kitzman

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PowerPoint slides

Glossary

Low-frequency variants

Single-nucleotide variants, insertions and deletions (indels) or copy-number variants that have minor allele frequency in a population <1%; that is, variants found on <1 out of every 100 haplotypes.

Private variants

Variants that are found in a single individual or pedigree and are thus recalcitrant to phasing by population-based methods owing to their absence from reference panels.

Linkage disequilibrium

A measure of the probability that two polymorphic loci do not segregate independently within a population.

Padlock probes

Single-stranded DNA oligonucleotides that have a constant region flanked by two targeting 'arms' that are complementary to the sequence of a genomic target. After highly specific hybridization to the target, the probes can be circularized and analysed for genotyping.

Personal genome

A substantially complete genome sequence of a single individual, typically obtained to attempt to describe or predict medical or other traits of that individual.

Mate-paired

Pertaining to a type of sequencing library preparation in which portions of a haplotype separated by 3–5 kb are brought into proximity by fragmentation and in vitro circularization. By sequencing across the junction of these circles, variants that are separated in genomic space but that appear on the same fragments can be jointly phased.

High-molecular-weight (HMW) genomic DNA

Genomic DNA isolated in such a way as to preserve long intact DNA fragments, ideally exceeding 100 kb on average. The ideal length may differ depending on the application.

Fosmids

DNA cloning vectors containing up to 40 kb of insert, typically packaged in bulk into phage and transfected into Escherichia coli, in which a library can be propagated.

Multiple displacement amplification

(MDA). A method for high-gain whole-genome amplification in which a low input mass of high-molecular-weight DNA is exponentially copied by random priming with short oligonucleotides, followed by primer extension with a strand-displacing polymerase at a constant temperature. Resulting amplicons are typically several kilobases in length.

Complete Genomics sequencing platform

A form of high-throughput short-read sequencing technology and a suite of analysis tools offered as a commercial service.

Illumina sequencing platform

The most commonly used form of high-throughput short-read sequencing that offers a low cost per base.

Moleculo system

A commercial library preparation and in silico method for reconstructing the sequence of a 6–10-kb fragment of DNA using short-read sequencing instruments.

Long-read sequencing methods

Sequencing technologies in which either raw or computationally assembled reads exceed 1 kb, such that each read has a greater probability of capturing two or more variants on a single haplotype. They are typically associated with a higher cost per base and a lower throughput than short-read technologies.

Subassembly

An in silico method for reconstructing the sequence of a DNA fragment that exceeds the maximum read length of the sequencing instrument. Molecules of ~500 bp are uniquely tagged, amplified, concatemerized and randomly fragmented. Short reads capturing the tag and a random portion of the original fragment can be jointly assembled to recover the full-length sequence.

Single-molecule real-time (SMRT) sequencing

A form of sequencing technology that directly interrogates individual molecules of DNA and thus does not require library amplification before sequencing.

Nanopore sequencing

A method for DNA sequencing in which small changes in electrical current are detected as sequential bases of a DNA polymer pass through a 1 nm transmembrane protein or solid-state pore. As single molecules of DNA can be sequenced directly, no library amplification step is required.

Chromatin interaction maps

Sets of measurements of the pairwise 3D spatial proximity of many non-adjacent regions of genomic DNA in a nucleus, as ascertained experimentally by crosslinking chromatin, ligating together fragments of DNA that are associated with the crosslinked proteins, and sequencing.

Phred-scale quality scores

A scoring system, originally developed for assigning confidence to individual base calls from sequencing instruments, in which an estimated error probability (P) is converted to a quality score (Q) by the transformation Q = −10 log10(P).

Runs of homozygosity

Regions of the genome above a given distance threshold at which both haplotypes are identical.

Compound heterozygosity

The presence of two different recessive alleles, one on each haplotype, in a specific gene in a single individual. It is particularly relevant for autosomal recessive genetic diseases, which are frequently caused by compound heterozygosity in non-consanguineous pedigrees.

Variant imputation

A statistically grounded method for 'filling in' missing alleles in sparsely genotyped individuals to increase the power of association studies on the basis of similarity to reference panels of previously ascertained haplotypes.

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Snyder, M., Adey, A., Kitzman, J. et al. Haplotype-resolved genome sequencing: experimental methods and applications. Nat Rev Genet 16, 344–358 (2015). https://doi.org/10.1038/nrg3903

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