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During meiotic recombination, homologue-templated repair of programmed DNA double-strand breaks (DSBs) produces relatively few crossovers and many difficult-to-detect non-crossovers. By intercrossing two diverged mouse subspecies over five generations and deep-sequencing 119 offspring, we detect thousands of crossover and non-crossover events genome-wide with unprecedented power and spatial resolution. We find that both crossovers and non-crossovers are strongly depleted at DSB hotspots where the DSB-positioning protein PRDM9 fails to bind to the unbroken homologous chromosome, revealing that PRDM9 also functions to promote homologue-templated repair. Our results show that complex non-crossovers are much rarer in mice than humans, consistent with complex events arising from accumulated non-programmed DNA damage. Unexpectedly, we also find that GC-biased gene conversion is restricted to non-crossover tracts containing only one mismatch. These results demonstrate that local genetic diversity profoundly alters meiotic repair pathway decisions via at least two distinct mechanisms, impacting genome evolution and Prdm9 -related hybrid infertility. During meiotic recombination, genetic information is transferred or exchanged between parental chromosome copies. Using a large hybrid mouse pedigree, the authors generated high-resolution maps of these transfer/exchange events and discovered new properties governing their processing and resolution.

PRDM9 binding localizes almost all meiotic recombination sites in humans and mice. However, most PRDM9-bound loci do not become recombination hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human PRDM9 binding sites in a transfected human cell line and measured PRDM9-induced histone modifications. These data reveal varied DNA-binding modalities of PRDM9. We also find that human PRDM9 frequently binds promoters, despite their low recombination rates, and it can activate expression of a small number of genes including CTCFL and VCX. Furthermore, we identify specific sequence motifs that predict consistent, localized meiotic recombination suppression around a subset of PRDM9 binding sites. These motifs strongly associate with KRAB-ZNF protein binding, TRIM28 recruitment, and specific histone modifications. Finally, we demonstrate that, in addition to binding DNA, PRDM9's zinc fingers also mediate its multimerization, and we show that a pair of highly diverged alleles preferentially form homo-multimers. Human cells have two copies of each chromosome: one from the mother, and one from the father. When cells divide to form sex cells, such as sperm or egg cells, the maternal and paternal chromosomes line up next to each other and swap some of their DNA. This process, known as genetic recombination, creates different versions of genes and ensures that we are all unique – or genetically diverse. Recombination is a complex process that is largely controlled by a protein called PRDM9. This protein binds DNA at particular spots on the chromosome and directs other proteins to carry out recombination nearby. However, not all of PRDM9’s binding sites are known, and not all regions that PRDM9 binds to undergo recombination. Until now, it was not understood why this is the case at fine scales. To investigate this further, Altemose et al. activated the human version of PRDM9 in human kidney cells grown in the laboratory. The results showed that PRDM9 often bound near the start sites of genes, although these regions rarely undergo recombination in humans. When PRDM9 bound near these sites, it sometimes turned the gene on, which suggests that it may also help to regulate the activity of genes. Moreover, a specific group of DNA-binding proteins, called KRAB-ZNF proteins, appear to suppress recombination wherever they bind, which explains why some PRDM9 binding sites do not recombine. Lastly, Altemose et al. discovered that the part of PRDM9 that binds to DNA can also bind to other copies of PRDM9 proteins. This self-binding ability might play a role in bringing together the maternal and paternal chromosomes at the correct spots during recombination. Together, these results shed new light on the recombination process, which is a driving force in the formation of new species and essential for fertility. A next step will be to study these results further in tissues of the reproductive organs. This will provide a better understanding of the forces that shape human evolution.

Although the past decade has seen tremendous progress in our understanding of fine-scale recombination, little is known about non-crossover (NCO) gene conversion. We report the first genome-wide study of NCO events in humans. Using SNP array data from 98 meioses, we identified 103 sites affected by NCO, of which 50/52 were confirmed in sequence data. Overlap with double strand break (DSB) hotspots indicates that most of the events are likely of meiotic origin. We estimate that a site is involved in a NCO at a rate of 5.9 × 10−6/bp/generation, consistent with sperm-typing studies, and infer that tract lengths span at least an order of magnitude. Observed NCO events show strong allelic bias at heterozygous AT/GC SNPs, with 68% (58–78%) transmitting GC alleles (p = 5 × 10−4). Strikingly, in 4 of 15 regions with resequencing data, multiple disjoint NCO tracts cluster in close proximity (∼20–30 kb), a phenomenon not previously seen in mammals. The genetic information inside our cells is stored in the form of chromosomes, which are carefully packaged strands of DNA. Most human cells contain a pair of each chromosome: one inherited from the mother and another from the father. Typically, when a human cell divides, it duplicates all of its chromosomes and then places one copy of each into the two new cells. However, a different process—known as ‘meiosis’—occurs when a human cell divides to make the cells involved in sexual reproduction (i.e., egg cells in females and sperm cells in males). First, the cell duplicates all of its chromosomes as before, but then it pairs the chromosomes originally from the mother with the equivalent chromosomes from the father. These paired chromosomes then swap sections of DNA. Next, the cell divides, and the resulting cells divide again; this produces four new cells that each contain a single, unique copy of every chromosome. In the process of swapping sections of DNA between chromosomes, the DNA molecule inside the chromosome is broken and different sections of DNA are then joined together. This can occur by one of two methods: ‘crossover events’ that produce a final chromosome made up of long sequences from each of the contributing chromosomes; and ‘non-crossover events’, where only a small section of DNA is swapped between the chromosomes. Research has tended to focus on DNA breaks and crossover events. Now, Williams et al. have looked at the genetic sequences transmitted by both parents to 49 humans—revealing information about a total of 98 meioses—and scoured them for evidence of non-crossover events. In addition to finding 103 sites where these events occurred, Williams et al. discovered that non-crossover events are more frequent around sites where crossover events also have a higher frequency. This suggests that the mechanism that initiates non-crossover events is shared with crossovers, and that non-crossover events primarily occur during meiosis. Unexpectedly, in some areas non-crossover events were found close to each other in ‘clusters’, which had not previously been seen in humans. Non-crossover events will only produce an observable change if the chromosomes involved have differences in the sequence of the DNA section that is swapped between them. The number of such variable genetic positions that non-crossover events affect in a generation is roughly the same number as the number of newly generated random mutations to the DNA sequence in a generation. Examining the DNA sequences transferred during non-crossover events also shows that two different types of DNA bases (cytosine and guanine) are more likely to be transmitted by a non-crossover event than are the other two bases (adenine and thymine). This bias indicates that non-crossover events are an important factor in driving genome evolution. In the future, sequencing the entire genome—the total genetic material—of many different people could provide further insights into non-crossover events in humans.

The largest gaps in the human genome assembly correspond to multi-megabase heterochromatic regions composed primarily of two related families of tandem repeats, Human Satellites 2 and 3 (HSat2,3). The abundance of repetitive DNA in these regions challenges standard mapping and assembly algorithms, and as a result, the sequence composition and potential biological functions of these regions remain largely unexplored. Furthermore, existing genomic tools designed to predict consensus-based descriptions of repeat families cannot be readily applied to complex satellite repeats such as HSat2,3, which lack a consistent repeat unit reference sequence. Here we present an alignment-free method to characterize complex satellites using whole-genome shotgun read datasets. Utilizing this approach, we classify HSat2,3 sequences into fourteen subfamilies and predict their chromosomal distributions, resulting in a comprehensive satellite reference database to further enable genomic studies of heterochromatic regions. We also identify 1.3 Mb of non-repetitive sequence interspersed with HSat2,3 across 17 unmapped assembly scaffolds, including eight annotated gene predictions. Finally, we apply our satellite reference database to high-throughput sequence data from 396 males to estimate array size variation of the predominant HSat3 array on the Y chromosome, confirming that satellite array sizes can vary between individuals over an order of magnitude (7 to 98 Mb) and further demonstrating that array sizes are distributed differently within distinct Y haplogroups. In summary, we present a novel framework for generating initial reference databases for unassembled genomic regions enriched with complex satellite DNA, and we further demonstrate the utility of these reference databases for studying patterns of sequence variation within human populations.

The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome.

Epidemiological studies have suggested differences in the rate of multiple sclerosis (MS) in individuals of European ancestry compared to African ancestry, motivating genetic scans to identify variants that could contribute to such patterns. In a whole-genome scan in 899 African-American cases and 1155 African-American controls, we confirm that African-Americans who inherit segments of the genome of European ancestry at a chromosome 1 locus are at increased risk for MS [logarithm of odds (LOD) = 9.8], although the signal weakens when adding an additional 406 cases, reflecting heterogeneity in the two sets of cases [logarithm of odds (LOD) = 2.7]. The association in the 899 individuals can be fully explained by two variants previously associated with MS in European ancestry individuals. These variants tag a MS susceptibility haplotype associated with decreased CD58 gene expression (odds ratio of 1.37; frequency of 84% in Europeans and 22% in West Africans for the tagging variant) as well as another haplotype near the FCRL3 gene (odds ratio of 1.07; frequency of 49% in Europeans and 8% in West Africans). Controlling for all other genetic and environmental factors, the two variants predict a 1.44-fold higher rate of MS in European-Americans compared to African-Americans.

Steven McCarroll, Giulio Genovese and colleagues report a new approach to help complete maps of the human genome reference sequence. They use a population-based admixture mapping approach and report the successful mapping of ~4 Mb of unplaced human euchromatic sequences. Tens of millions of base pairs of euchromatic human genome sequence, including many protein-coding genes, have no known location in the human genome. We describe an approach for localizing the human genome's missing pieces using the patterns of genome sequence variation created by population admixture. We mapped the locations of 70 scaffolds spanning 4 million base pairs of the human genome's unplaced euchromatic sequence, including more than a dozen protein-coding genes, and identified 8 new large interchromosomal segmental duplications. We find that most of these sequences are hidden in the genome's heterochromatin, particularly its pericentromeric regions. Many cryptic, pericentromeric genes are expressed at the RNA level and have been maintained intact for millions of years while their expression patterns diverged from those of paralogous genes elsewhere in the genome. We describe how knowledge of the locations of these sequences can inform disease association and genome biology studies.

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure that includes long palindromes, tandem repeats and segmental duplications 1 – 3 . As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished 4 , 5 . Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029-base-pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, showing the complete ampliconic structures of gene families TSPY , DAZ and RBMY ; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a previous assembly of the CHM13 genome 4 and mapped available population variation, clinical variants and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes. We present the complete 62,460,029-base-pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference.

Studies of genome regulation routinely use high-throughput DNA sequencing approaches to determine where specific proteins interact with DNA, and they rely on DNA amplification and short-read sequencing, limiting their quantitative application in complex genomic regions. To address these limitations, we developed directed methylation with long-read sequencing (DiMeLo-seq), which uses antibody-tethered enzymes to methylate DNA near a target protein’s binding sites in situ. These exogenous methylation marks are then detected simultaneously with endogenous CpG methylation on unamplified DNA using long-read, single-molecule sequencing technologies. We optimized and benchmarked DiMeLo-seq by mapping chromatin-binding proteins and histone modifications across the human genome. Furthermore, we identified where centromere protein A localizes within highly repetitive regions that were unmappable with short sequencing reads, and we estimated the density of centromere protein A molecules along single chromatin fibers. DiMeLo-seq is a versatile method that provides multimodal, genome-wide information for investigating protein–DNA interactions. DiMeLo-seq uses native long-read sequencing to examine protein–DNA interactions by mapping exogenous methylation marks generated by a nonspecific DNA methyltransferase, as well as profile endogenous CpG methylation simultaneously.