DNA sequencing decodes the millions of chemical letters that make up an organism’s genetic blueprint. This technology has revolutionized modern biology, allowing researchers to read the entire length of the genome with increasing speed and accuracy. Standard sequencing often breaks long DNA strands into small pieces before reading them. When reassembled, the resulting genetic picture is a blended consensus of information, obscuring the fundamental separation of genetic information inherited from each parent.
Understanding the Haplotype: The Problem Phased Sequencing Solves
Humans and many other organisms are diploid, possessing two complete sets of chromosomes in nearly every cell, one inherited from the mother and one from the father. While each chromosome pair contains the same genes, the specific variants, or alleles, often differ between the two copies. Capturing this distinction between the two parental copies is technically challenging with conventional sequencing.
The specific combination of alleles found along a single, continuous stretch of one parental chromosome is known as a haplotype. A person thus has two haplotypes for any given genomic region: the maternal copy and the paternal copy. Standard sequencing methods identify single-nucleotide variants (SNVs), but they cannot determine which variants sit together on the same chromosome copy. Instead, the results report a genotype, showing that two different variants exist but failing to specify if they are physically linked or separated across the two parental chromosomes.
This loss of linkage information is a limitation, especially when two variable sites are far apart, often exceeding the length of a typical sequencing read. For example, if sequencing shows both Variant A and Variant B are present, scientists cannot tell if the detrimental versions are paired on the same chromosome or separated. Phased sequencing, or genome phasing, resolves this ambiguity by separating the genetic data into its distinct parental haplotypes, revealing the true physical grouping of variants.
Technological Approaches to Determining Phased Sequences
Determining phased sequences requires several distinct technological and computational approaches. One direct and effective method involves physically observing long stretches of DNA containing multiple variants. Long-read sequencing technologies, such as those developed by PacBio or Oxford Nanopore, generate much longer DNA fragments than traditional methods. These extended reads can span hundreds of thousands of base pairs, often encompassing several variable sites.
When a single long read covers two or more heterozygous variants, it physically links them, unambiguously confirming they are located on the same parental chromosome. Highly accurate long reads, often called HiFi reads, offer the precision needed to detect subtle changes combined with the length necessary to connect them across long distances. This direct observation method generates long, continuous haplotype blocks for a single individual without relying on external information.
Alternatively, phasing can be achieved indirectly using computational methods that leverage family data, commonly known as trio analysis. Here, the DNA of a child is sequenced along with that of both biological parents. By comparing the child’s variants to the parents’, researchers assign each of the child’s alleles to the specific parental chromosome from which it was inherited. Any variant present only in the mother must be on the maternal chromosome, allowing the construction of the two distinct haplotypes.
A third set of methods uses statistical and population-level data to infer phase without requiring long reads or parental information. Statistical phasing relies on large reference panels of already-phased genomes to identify common variation patterns. This method works because variants located close together tend to be inherited together over many generations due to infrequent recombination, a phenomenon known as linkage disequilibrium. Computational algorithms use this frequency data to predict the most likely parental arrangement of variants for a new individual.
Impact and Real-World Applications of Phasing
Phased sequencing has transformed genetic research and personalized medicine by providing a higher-resolution view of the genome. In disease association studies, phasing is necessary because many complex diseases are caused not by a single variant, but by a specific combination of variants, or a unique haplotype. Accurately mapping these disease-associated haplotypes is necessary to pinpoint the actual causative region on the chromosome.
Phasing is also necessary for accurately identifying compound heterozygosity, where an individual carries two different disease-causing mutations in the same gene. If the mutations are separated across the maternal and paternal chromosomes, the individual may develop a recessive disorder. However, if both mutations are on the same chromosome, the individual will only be a carrier. Phased data provides the definitive answer to this distinction, which is necessary for accurate diagnosis and genetic counseling.
In pharmacogenomics, which studies how genetics affects drug response, phased data is especially informative. The efficacy or toxicity of many medications depends on specific combinations of alleles within drug-metabolizing genes. Knowing the precise HLA haplotype, a highly variable region of the genome, can help predict severe adverse drug reactions. This makes phasing a necessary step toward personalized medicine and drug dosing.