Human-genome analysis has rapidly progressed from determining the reference sequence for the ‘average’ human genome to prolific sequencing of personal genomes, and it may seem surprising that single-cell approaches have anything more to contribute. However, some aspects of the human genome have been very challenging to determine using conventional techniques. For example, all of us have two genomes within each of our cells—one from our mother and another from our father—and the location of sequence variation in each haploid genome can have a significant effect on gene expression, protein function and disease.
The best-known example of this is variation in human leukocyte antigen (HLA) genes, whose haplotype is important to understand for bone marrow transplants, but it applies just as well to compound heterozygous mutations—two mutations at a single locus that may be harmless when they reside in the same haplotype, but deleterious when distributed between the maternal and paternal allele. Current techniques have not been able to resolve these differences, known as haplotype determination, at a genome-wide level with any degree of precision. The best conventional methods for haplotype determination require additional sequencing within a family pedigree, mainly from parents. Clearly, this is not a practical approach in most clinical situations.
Single-cell chromosome isolation enabled the first genome-wide haplotype measurement, with haplotype phase determined across the lengths of entire chromosomes10
. This work was quickly followed by related single-cell sequencing approaches using small numbers of cells11
, or in the case of males, large numbers of individual sperm cells12
. We expect that further application of these techniques and long-read sequencing technologies that determine haplotypes of genomic segments will accelerate analysis of refractory parts of the human genome. The HLA region, which is the most polymorphic part of the human genome and intimately involved in the immune system and many aspects of human health, is a particularly interesting target, but up to now it has been sequenced in only a limited number of individuals owing to its haplotype complexity.
Another area touched by single-cell genomics is the analysis of recombination patterns across human diversity. Recombination is the cutting and pasting of large blocks of the maternally and paternally inherited chromosomes to create entirely new genomes in sperm and egg cells, and it is a major contributor to genetic diversity in the human species. It is known that recombination does not happen with uniform probability across the genome; rather, certain ‘hot spots’ experience frequent recombination. One of the earliest contributions of single-cell genomics was to show that there is differential hot-spot usage between individuals: some spots may be hot for one person but not another13
. More recently, single-cell approaches have enabled the measurement of genome-wide recombination patterns and mutation rates in individual sperm cells, enabling the first studies of genome-wide hot-spot behavior within individuals12,14
. We expect that further genomic analyses of single sperm cells will enable the study of recombination mutants (for example, in individuals carrying rare alleles of PRDM9
) as well as the potential diagnosis of those with meiotic dysfunction related to sterility and infertility.