Massively parallel sequencing and capillary electrophoresis of a novel panel of falcon STRs: Concordance with minisatellite DNA profiles from historical wildlife crime.

Birds of prey have suffered persecution for centuries through trapping, shooting, poisoning and theft from the wild to meet the demand from egg collectors and falconers; they were also amongst the earliest beneficiaries of DNA testing in wildlife forensics. Here we report the identification and characterisation of 14 novel tetramer, pentamer and hexamer short tandem repeat (STR) markers which can be typed either by capillary electrophoresis or massively parallel sequencing (MPS) and apply them to historical casework samples involving 49 peregrine falcons, 30 of which were claimed to be the captively bred offspring of nine pairs. The birds were initially tested in 1994 with a multilocus DNA fingerprinting probe, a sex test and eight single-locus minisatellite probes (SLPs) demonstrating that 23 birds were unrelated to the claimed parents. The multilocus and SLP approaches were highly discriminating but extremely time consuming and required microgram quantities of high molecular weight DNA and the use of radioisotopes. The STR markers displayed between 2 and 21 alleles per locus (mean = 7.6), lengths between 140 and 360 bp, and heterozygosities from 0.4 to 0.93. They produced wholly concordant conclusions with similar discrimination power but in a fraction of the time using a hundred-fold less DNA and with standard forensic equipment. Furthermore, eleven of these STRs were amplified in a single reaction and typed using MPS on the Illumina MiSeq platform revealing eight additional alleles (three with variant repeat structures and five solely due to flanking SNPs) across four loci. This approach gave a random match probability of < 1E-9, and a parental pair false inclusion probability of < 1E-5, with a further ten-fold reduction in the amount of DNA required (~3 ng) and the potential to analyse mixed samples. These STRs will be of value in monitoring wild populations of these key indicator species as well as for testing captive breeding claims and establishing a database of captive raptors. They have the potential to resolve complex cases involving trace, mixed and degraded samples from raptor persecution casework representing a significant advance over the previously applied methods.

The Genomic Impact of European Colonization of the Americas.

The human genetic diversity of the Americas has been affected by several events of gene flow that have continued since the colonial era and the Atlantic slave trade. Moreover, multiple waves of migration followed by local admixture occurred in the last two centuries, the impact of which has been largely unexplored. Here, we compiled a genome-wide dataset of ∼12,000 individuals from twelve American countries and ∼6,000 individuals from worldwide populations and applied haplotype-based methods to investigate how historical movements from outside the New World affected (1) the genetic structure, (2) the admixture profile, (3) the demographic history, and (4) sex-biased gene-flow dynamics of the Americas. We revealed a high degree of complexity underlying the genetic contribution of European and African populations in North and South America, from both geographic and temporal perspectives, identifying previously unreported sources related to Italy, the Middle East, and to specific regions of Africa.

Recombination hotspots in an extended human pseudoautosomal domain predicted from double-strand break maps and characterized by sperm-based crossover analysis.

The human X and Y chromosomes are heteromorphic but share a region of homology at the tips of their short arms, pseudoautosomal region 1 (PAR1), that supports obligate crossover in male meiosis. Although the boundary between pseudoautosomal and sex-specific DNA has traditionally been regarded as conserved among primates, it was recently discovered that the boundary position varies among human males, due to a translocation of ~110 kb from the X to the Y chromosome that creates an extended PAR1 (ePAR). This event has occurred at least twice in human evolution. So far, only limited evidence has been presented to suggest this extension is recombinationally active. Here, we sought direct proof by examining thousands of gametes from each of two ePAR-carrying men, for two subregions chosen on the basis of previously published male X-chromosomal meiotic double-strand break (DSB) maps. Crossover activity comparable to that seen at autosomal hotspots was observed between the X and the ePAR borne on the Y chromosome both at a distal and a proximal site within the 110-kb extension. Other hallmarks of classic recombination hotspots included evidence of transmission distortion and GC-biased gene conversion. We observed good correspondence between the male DSB clusters and historical recombination activity of this region in the X chromosomes of females, as ascertained from linkage disequilibrium analysis; this suggests that this region is similarly primed for crossover in both male and female germlines, although sex-specific differences may also exist. Extensive resequencing and inference of ePAR haplotypes, placed in the framework of the Y phylogeny as ascertained by both Y microsatellites and single nucleotide polymorphisms, allowed us to estimate a minimum rate of crossover over the entire ePAR region of 6-fold greater than genome average, comparable with pedigree estimates of PAR1 activity generally. We conclude ePAR very likely contributes to the critical crossover function of PAR1.

Transmission distortion affecting human noncrossover but not crossover recombination: a hidden source of meiotic drive.

Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simultaneously analysed both types of recombinant molecule in sperm DNA at six highly active hotspots, and looked for disparity in the transmission of allelic variants indicative of any cis-acting influences. At two of the hotspots we identified a novel form of biased transmission that was exclusive to the noncrossover class of recombinant, and which presumably arises through differences between crossovers and noncrossovers in heteroduplex formation and biased mismatch repair. This form of biased gene conversion is not predicted to influence hotspot activity as previously noted for SNPs that affect recombination initiation, but does constitute a powerful and previously undetected source of recombination-driven meiotic drive that by extrapolation may affect thousands of recombination hotspots throughout the human genome. Intriguingly, at both of the hotspots described here, this drive favours strong (G/C) over weak (A/T) base pairs as might be predicted from the well-established correlations between high GC content and recombination activity in mammalian genomes.

A major recombination hotspot in the XqYq pseudoautosomal region gives new insight into processing of human gene conversion events.

Recombination plays a fundamental role in meiosis. Non-exchange gene conversion (non-crossover, NCO) may facilitate homologue pairing, while reciprocal crossover (CO) physically connects homologues so they orientate appropriately on the meiotic spindle. In males, X-Y homologous pairing and exchange occurs within the two pseudoautosomal regions (PARs) together comprising <5% of the human sex chromosomes. Successful meiosis depends on an obligatory CO within PAR1, while the nature and role of exchange within PAR2 is unclear. Here, we describe the identification and characterization of a typical ~1 kb wide recombination hotspot within PAR2. We find that both COs and NCOs are strongly modulated in trans by the presumed chromatin remodelling protein PRDM9, and in cis by a single nucleotide polymorphism (SNP) located at the hotspot centre that appears to influence recombination initiation and which causes biased gene conversion in SNP heterozygotes. This, the largest survey to date of human NCOs reveals for the first time substantial inter-individual variation in the NCO:CO ratio. Although the extent of biased transmission at the central marker in COs is similar across men, it is highly variable among NCO recombinants. This suggests that cis-effects are mediated not only through recombination initiation frequencies varying between haplotypes but also through subsequent processing, with the potential to significantly intensify meiotic drive of hotspot-suppressing alleles. The NCO:CO ratio and extent of transmission distortion among NCOs appear to be inter-related, suggesting the existence of two NCO pathways in humans.

PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans.

PRDM9 has recently been identified as a likely trans regulator of meiotic recombination hot spots in humans and mice. PRDM9 contains a zinc finger array that, in humans, can recognize a short sequence motif associated with hot spots, with binding to this motif possibly triggering hot-spot activity via chromatin remodeling. We now report that human genetic variation at the PRDM9 locus has a strong effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Subtle changes within the zinc finger array can create hot-spot nonactivating or enhancing variants and can even trigger the appearance of a new hot spot, suggesting that PRDM9 is a major global regulator of hot spots in humans. Variation at the PRDM9 locus also influences aspects of genome instability-specifically, a megabase-scale rearrangement underlying two genomic disorders as well as minisatellite instability-implicating PRDM9 as a risk factor for some pathological genome rearrangements.

Analysis of meiotic recombination products from human sperm.

Traditional methods for surveying meiotic recombination in humans are limited to pedigree and linkage disequilibrium analyses. We have developed assays that allow the direct detection of crossover and gene conversion molecules in batches of sperm DNA. To date, we have characterized 26 recombination hotspots by allele-specific PCR and selectively amplified recombinant DNA molecules from these regions. These analyses have revealed that meiotic crossover hotspots in humans are highly localized and flanked by DNA segments where recombination is suppressed. The centers of crossover hotspots are also active in noncrossover recombination, displaying short conversion tracts.

Complex germline and somatic mutation processes at a haploid human minisatellite shown by single-molecule analysis.

Mutation at most human minisatellites is driven by complex interallelic processes that give rise to a high degree of length polymorphism and internal structural variation. MSY1, the only highly variable minisatellite on the non-recombining region of the Y chromosome, is constitutively haploid and therefore precluded from interallelic interactions, yet maintains high diversity in both length and structure. To investigate the basis of its mutation processes, an unbiased structural analysis of >500 single-molecule MSY1 PCR products from matched sperm and blood samples from a single donor was undertaken. The overall mutation frequencies in sperm and blood DNAs were not significantly different, at 2.68% and 1.88%, respectively. Sperm DNA showed significantly more length mutants than blood DNA, with mutants in both tissues involving small-scale (1-3 repeat units in a 77 repeat progenitor allele) increases or decreases in repeat block lengths, with no gain or loss bias. Isometric mutations altering structure but not length were found in both tissues, and involved either the apparent shift of a boundary between repeat unit blocks (a 'boundary switch') or the conversion of a repeat within a block to a different repeat type ('modular structure' mutant). There was a significant excess of boundary switch mutants and deficit of modular structure mutants in sperm. A comparison of mutant structures with phylogenetically matched alleles in population samples showed that alleles with structures resembling the blood mutants were unlikely to arise in populations. Mutation seems likely to involve gene conversion via synthesis-dependent strand annealing, and the blood-sperm differences may reflect more relaxed constraint on sister chromatid alignment in blood.

Meiotic recombination hot spots and human DNA diversity.

Meiotic recombination plays a key role in the maintenance of sequence diversity in the human genome. However, little is known about the fine-scale distribution and processes of recombination in human chromosomes, or how these impact on patterns of human diversity. We have therefore developed sperm typing systems that allow human recombination to be analysed at very high resolution. The emerging picture is that human crossovers are far from randomly distributed but instead are targeted into very narrow hot spots that can profoundly influence patterns of haplotype diversity in the human genome. These hot spots provide fundamental information on processes of human crossover and gene conversion, as well as evidence that they can violate basic rules of Mendelian inheritance.

Intense and highly localized gene conversion activity in human meiotic crossover hot spots.

Meiotic gene conversion has an important role in allele diversification and in the homogenization of gene and other repeat DNA sequence families, sometimes with pathological consequences. But little is known about the dynamics of gene conversion in humans and its relationship to meiotic crossover. We therefore developed screening and selection methods to characterize sperm conversions in two meiotic crossover hot spots in the major histocompatibility complex (MHC) and one in the sex chromosomal pseudoautosomal pairing region PAR1 (ref. 9). All three hot spots are active in gene conversion and crossover. Conversion tracts are short and define a steep bidirectional gradient centered at the peak of crossover activity, consistent with crossovers and conversions being produced by the same recombination-initiating events. These initiations seem to be spread over a narrow zone, rather than occurring at a single site, and seem preferentially to yield conversions rather than crossovers. Crossover breakpoints are more broadly diffused than conversion breakpoints, suggesting either differences between conversion and crossover processing after initiation or the existence of a quality control checkpoint at which short interactions between homologous chromosomes are preferentially aborted as conversions.

DNA enrichment by allele-specific hybridization (DEASH): a novel method for haplotyping and for detecting low-frequency base substitutional variants and recombinant DNA molecules.

Detecting rare sequence variants in genomic DNA is central to the analysis of de novo mutation and recombination events and the detection of rare pathological mutations in mixed cell populations. Current PCR techniques suffer from noise that limits detection to variants present at a frequency of at least 10(-4)-10(-5) per cell. We now describe an alternative approach that recovers genomic DNA molecules containing a known single-nucleotide variant by hybridization selection using a biotinylated allele-specific oligonucleotide, followed by hybrid capture on streptavidin-coated paramagnetic beads and subsequent analysis by PCR. This technique of DNA enrichment by allele-specific hybridization (DEASH) is fast, effective for all tested single-nucleotide polymorphisms (SNPs), and can recover large (>10 kb) single-stranded molecules. A single round of DEASH is effective in separating haplotypes from genomic DNA and can not only readily detect and validate DNA molecules containing a single base change at a frequency of 10(-5) per cell, but can also place these changes within the context of an extended haplotype. This technique offers a new approach to the analysis of mutation and recombination, and has the potential to detect very rare de novo base substitutions.

Crossover clustering and rapid decay of linkage disequilibrium in the Xp/Yp pseudoautosomal gene SHOX.

Crossover between the human sex chromosomes during male meiosis is restricted to the terminal pseudoautosomal pairing regions. An obligatory exchange occurs in PAR1, an Xp/Yp pseudoautosomal region of 2.6 Mb, which creates a male-specific recombination 'hot domain' with a recombination rate that is about 20 times higher than the genome average. Low-resolution analysis of PAR1 suggests that crossovers are distributed fairly randomly. By contrast, linkage disequilibrium (LD) and sperm crossover analyses indicate that crossovers in autosomal regions tend to cluster into 'hot spots' of 1-2 kb that lie between islands of disequilibrium of tens to hundreds of kilobases. To determine whether at high resolution this autosomal pattern also applies to PAR1, we have examined linkage disequilibrium over an interval of 43 kb around the gene SHOX. Here we show that in northern European populations, disequilibrium decays rapidly with physical distance, which is consistent with this interval of PAR1 being recombinationally active in male meiosis. Analysis of a subregion of 9.9 kb in sperm shows, however, that crossovers are not distributed randomly, but instead cluster into an intense recombination hot spot that is very similar in morphology to autosomal hot spots. Thus, PAR1 crossover activity may be influenced by male-specific hot spots that are highly suitable for characterization by sperm DNA analysis.