Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

Correlation between genetic and geographic structure in Europe.

Understanding the genetic structure of the European population is important, not only from a historical perspective, but also for the appropriate design and interpretation of genetic epidemiological studies. Previous population genetic analyses with autosomal markers in Europe either had a wide geographic but narrow genomic coverage [1, 2], or vice versa [3-6]. We therefore investigated Affymetrix GeneChip 500K genotype data from 2,514 individuals belonging to 23 different subpopulations, widely spread over Europe. Although we found only a low level of genetic differentiation between subpopulations, the existing differences were characterized by a strong continent-wide correlation between geographic and genetic distance. Furthermore, mean heterozygosity was larger, and mean linkage disequilibrium smaller, in southern as compared to northern Europe. Both parameters clearly showed a clinal distribution that provided evidence for a spatial continuity of genetic diversity in Europe. Our comprehensive genetic data are thus compatible with expectations based upon European population history, including the hypotheses of a south-north expansion and/or a larger effective population size in southern than in northern Europe. By including the widely used CEPH from Utah (CEU) samples into our analysis, we could show that these individuals represent northern and western Europeans reasonably well, thereby confirming their assumed regional ancestry.

High frequency of lactose intolerance in a prehistoric hunter-gatherer population in northern Europe.

BACKGROUND: Genes and culture are believed to interact, but it has been difficult to find direct evidence for the process. One candidate example that has been put forward is lactase persistence in adulthood, i.e. the ability to continue digesting the milk sugar lactose after childhood, facilitating the consumption of raw milk. This genetic trait is believed to have evolved within a short time period and to be related with the emergence of sedentary agriculture. RESULTS: Here we investigate the frequency of an allele (-13910*T) associated with lactase persistence in a Neolithic Scandinavian population. From the 14 individuals originally examined, 10 yielded reliable results. We find that the T allele frequency was very low (5%) in this Middle Neolithic hunter-gatherer population, and that the frequency is dramatically different from the extant Swedish population (74%). CONCLUSIONS: We conclude that this difference in frequency could not have arisen by genetic drift and is either due to selection or, more likely, replacement of hunter-gatherer populations by sedentary agriculturalists.

Homogeneity in mitochondrial DNA control region sequences in Swedish subpopulations.

In order to promote mitochondrial DNA (mtDNA) testing in Sweden we have typed 296 Swedish males, which will serve as a Swedish mtDNA frequency database. The tested males were taken from seven geographically different regions representing the contemporary Swedish population. The complete mtDNA control region was typed and the Swedish population was shown to have high haplotype diversity with a random match probability of 0.5%. Almost 47% of the tested samples belonged to haplogroup H and further haplogroup comparison with worldwide populations clustered the Swedish mtDNA data together with other European populations. AMOVA analysis of the seven Swedish subregions displayed no significant maternal substructure in Sweden (F (ST) = 0.002). Our conclusion from this study is that the typed Swedish individuals serve as good representatives for a Swedish forensic mtDNA database. Some caution should, however, be taken for individuals from the northernmost part of Sweden (provinces of Norrbotten and Lapland) due to specific demographic conditions. Furthermore, our analysis of a small sample set of a Swedish Saami population confirmed earlier findings that the Swedish Saami population is an outlier among European populations.

Barking up the wrong tree: modern northern European dogs fail to explain their origin.

BACKGROUND: Geographic distribution of the genetic diversity in domestic animals, particularly mitochondrial DNA, has often been used to infer centers of domestication. The underlying presumption is that phylogeographic patterns among domesticates were established during, or shortly after the domestication. Human activities are assumed not to have altered the haplogroup frequencies to any great extent. We studied this hypothesis by analyzing 24 mtDNA sequences in ancient Scandinavian dogs. Breeds originating in northern Europe are characterized by having a high frequency of mtDNA sequences belonging to a haplogroup rare in other populations (HgD). This has been suggested to indicate a possible origin of the haplogroup (perhaps even a separate domestication) in central or northern Europe. RESULTS: The sequences observed in the ancient samples do not include the haplogroup indicative for northern European breeds (HgD). Instead, several of them correspond to haplogroups that are uncommon in the region today and that are supposed to have Asian origin. CONCLUSION: We find no evidence for local domestication. We conclude that interpretation of the processes responsible for current domestic haplogroup frequencies should be carried out with caution if based only on contemporary data. They do not only tell their own story, but also that of humans.

Y-chromosome diversity in Sweden - a long-time perspective.

Sixteen Y-chromosomal binary markers and nine Y-chromosome short tandem repeats were analyzed in a total of 383 unrelated males from seven different Swedish regions, one Finnish region and a Swedish Saami population in order to address questions about the origin and genetic structure of the present day population in Sweden. Haplogroup I1a* was found to be the most common haplogroup in Sweden and accounted, together with haplogroups R1b3, R1a1 and N3, for over 80% of the male lineages. Within Sweden, a minor stratification was found in which the northern region Västerbotten differed significantly (P < 0.05) from the other Swedish regions. A flow of N3 chromosomes into Västerbotten mainly from Saami and Finnish populations could be one explanation for this stratification. However, the demographic history of Västerbotten involving a significant male absence during the 17th Century may also have had a large impact. Immigration of young men from elsewhere to Värmland at the same time, can be responsible for a similar deviation with I1a* haplotypes. Y chromosomes within haplogroup R1b3 were found to have the highest STR variation among all haplogroups and could thus be considered to be one of the earliest major male lineages present in Sweden. Regional haplotype variation, within R1b3, also showed a difference between two regions in the south of Sweden. This can also be traced from historical time and is visible in archaeological material. Overall this Y chromosome study provides interesting information about the genetic patterns and demographic events in the Swedish population.

Y-chromosome STR haplotypes in Sweden.

A total of 708 men, with Swedish names, from different parts of Sweden have been typed for the Y-chromosome minimal haplotype STR markers DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393 and DYS385. Of these, 403 men were of geographically undefined Swedish origin and the rest, 305, from seven defined geographical regions. PCR-products were detected by ABI377 using sequenced allelic ladders. An evaluation of the 708 chromosomes revealed 423 different haplotypes. Only 100 of the haplotypes were found more than once. The over all haplotype diversity was 0.994. The haplotype 14, 12, 28, 23, 10, 11, 13, 14-14 has the highest frequency of 5.79% and is significantly Swedish, when compared to other European populations.

Ancient mitochondrial DNA from the northern fringe of the Neolithic farming expansion in Europe sheds light on the dispersion process.

The European Neolithization process started around 12 000 years ago in the Near East. The introduction of agriculture spread north and west throughout Europe and a key question has been if this was brought about by migrating individuals, by an exchange of ideas or a by a mixture of these. The earliest farming evidence in Scandinavia is found within the Funnel Beaker Culture complex (Trichterbecherkultur, TRB) which represents the northernmost extension of Neolithic farmers in Europe. The TRB coexisted for almost a millennium with hunter-gatherers of the Pitted Ware Cultural complex (PWC). If migration was a substantial part of the Neolithization, even the northerly TRB community would display a closer genetic affinity to other farmer populations than to hunter-gatherer populations. We deep-sequenced the mitochondrial hypervariable region 1 from seven farmers (six TRB and one Battle Axe complex, BAC) and 13 hunter-gatherers (PWC) and authenticated the sequences using postmortem DNA damage patterns. A comparison with 124 previously published sequences from prehistoric Europe shows that the TRB individuals share a close affinity to Central European farmer populations, and that they are distinct from hunter-gatherer groups, including the geographically close and partially contemporary PWC that show a close affinity to the European Mesolithic hunter-gatherers.

A universal method for species identification of mammals utilizing next generation sequencing for the analysis of DNA mixtures.

Species identification can be interesting in a wide range of areas, for example, in forensic applications, food monitoring and in archeology. The vast majority of existing DNA typing methods developed for species determination, mainly focuses on a single species source. There are, however, many instances where all species from mixed sources need to be determined, even when the species in minority constitutes less than 1 % of the sample. The introduction of next generation sequencing opens new possibilities for such challenging samples. In this study we present a universal deep sequencing method using 454 GS Junior sequencing of a target on the mitochondrial gene 16S rRNA. The method was designed through phylogenetic analyses of DNA reference sequences from more than 300 mammal species. Experiments were performed on artificial species-species mixture samples in order to verify the method's robustness and its ability to detect all species within a mixture. The method was also tested on samples from authentic forensic casework. The results showed to be promising, discriminating over 99.9 % of mammal species and the ability to detect multiple donors within a mixture and also to detect minor components as low as 1 % of a mixed sample.

Analysis of radiocarbon, stable isotopes and DNA in teeth to facilitate identification of unknown decedents.

The characterization of unidentified bodies or suspected human remains is a frequent and important task for forensic investigators. However, any identification method requires clues to the person's identity to allow for comparisons with missing persons. If such clues are lacking, information about the year of birth, sex and geographic origin of the victim, is particularly helpful to aid in the identification casework and limit the search for possible matches. We present here results of stable isotope analysis of (13)C and (18)O, and bomb-pulse (14)C analyses that can help in the casework. The (14)C analysis of enamel provided information of the year of birth with an average absolute error of 1.8±1.3 years. We also found that analysis of enamel and root from the same tooth can be used to determine if the (14)C values match the rising or falling part of the bomb-curve. Enamel laydown times can be used to estimate the date of birth of individuals, but here we show that this detour is unnecessary when using a large set of crude (14)C data of tooth enamel as a reference. The levels of (13)C in tooth enamel were higher in North America than in teeth from Europe and Asia, and Mexican teeth showed even higher levels than those from USA. DNA analysis was performed on 28 teeth, and provided individual-specific profiles in most cases and sex determination in all cases. In conclusion, these analyses can dramatically limit the number of possible matches and hence facilitate person identification work.

DNA microarray as a tool in establishing genetic relatedness--Current status and future prospects.

In the past decades, microarray technology has definitely put an edge to the field of genetic research. Our aim was to determine whether single nucleotide polymorphism (SNP) microarrays could be used as a tool in establishing genetic relationships where current molecular genetic methods are not sufficient. We used the Genechip, Affymetrix GenomeWide SNP Array 6.0, which detects more than 900,000 SNP markers dispersed throughout the human genome. The intention was to find a good selection of SNP markers that could be used for statistical evaluation of relatedness in a forensic setting. We conducted pairwise comparisons in the R-package FEST as well as pedigree comparisons in Merlin. Our methods were applied on two separate families, where relationships as distant as 3rd cousins were known. In addition, a question about a possible common ancestry between the two families was tested. Relationships as distant as 2nd cousins could be readily distinguished both from unrelated and other, genetically, closer relationships. This was achieved with a selection of 5774 markers, where each pair of markers was separated by a genetic distance of at least 0.5cM (centiMorgan). When considering 3rd cousins, and more distant relationships, the number of markers needs to be extended, consequently decreasing the genetic distance between the markers. However, inclusion of a too large number of markers presents new challenges and our results imply that the use of too dense sets of markers always yields the highest probability for the genetically closest relationship hypothesis. Simulations confirm that this is most probably caused by the fact that the computational model assumes linkage equilibrium between markers, a problem that will be further evaluated. Our results do however suggest that SNP-data derived from microarrays are well suited for kinship determination provided linkage disequilibrium is properly accounted for.

Finding the founder of Stockholm - a kinship study based on Y-chromosomal, autosomal and mitochondrial DNA.

Historical records claim that Birger Magnusson (died 1266), famous regent of Sweden and the founder of Stockholm, was buried in Varnhem Abbey in Västergötland. After being lost for centuries, his putative grave was rediscovered during restoration work in the 1920s. Morphological analyses of the three individuals in the grave concluded that the older male, the female and the younger male found in the grave were likely to be Birger, his second wife Mechtild of Holstein and his son Erik from a previous marriage. More recent evaluations of the data from the 1920s seriously questioned these conclusions, ultimately leading to the reopening and reexamination of the grave in 2002. Ancient DNA-analyses were performed to investigate if the relationship between the three individuals matched what we would expect if the individuals were Birger, Erik and Mechtild. We used pyrosequencing of Y-chromosomal and autosomal SNPs and compared the results with haplogroup frequencies of modern Swedes to investigate paternal relations. Possible maternal kinship was investigated by deep FLX-sequencing of overlapping mtDNA amplicons. The authenticity of the sequences was examined using data from independent extractions, massive clonal data, the c-statistics, and real-time quantitative data. We show that the males carry the same Y-chromosomal haplogroup and thus we cannot reject a father-son type of relation. Further, as shown by the mtDNA analyses, none of the individuals are maternally related. We conclude that the graves indeed belong to Birger, Erik and Mechtild, or to three individuals with the exact same kind of biological relatedness.

Using X-chromosomal markers in relationship testing: calculation of likelihood ratios taking both linkage and linkage disequilibrium into account.

X-chromosomal markers in forensic genetics have become more widely used during recent years, particularly for relationship testing. Linkage and linkage disequilibrium (LD) must typically be accounted for when using close X-chromosomal markers. Thus, when producing the weight-of-evidence, given by a DNA-analysis with markers that are linked, the normally used product rule is invalid. Here we present an implementation of an efficient model for calculating likelihood ratios (LRs) with markers on the X-chromosome which are linked and in LD. Furthermore, the model was applied on several cases based on data from the eight X-chromosomal loci included in the Mentype(®) Argus X-8 (Biotype). Using a simulation approach we showed that the use of X-chromosome data can offer valuable information for choosing between the alternatives in each of the cases we studied, and that the LR can be high in several cases. We demonstrated that when linkage and LD were disregarded, as opposed to taken into account, the difference in calculated LRs could be considerable. When these differences were large, the estimated haplotype frequencies often had a strong impact and we present a method to estimate haplotype frequencies. Our conclusion is that linkage and LD should be accounted for when using the tested set of markers, and the used model is an efficient way of doing so.

Ancient DNA reveals lack of continuity between neolithic hunter-gatherers and contemporary Scandinavians.

The driving force behind the transition from a foraging to a farming lifestyle in prehistoric Europe (Neolithization) has been debated for more than a century [1-3]. Of particular interest is whether population replacement or cultural exchange was responsible [3-5]. Scandinavia holds a unique place in this debate, for it maintained one of the last major hunter-gatherer complexes in Neolithic Europe, the Pitted Ware culture [6]. Intriguingly, these late hunter-gatherers existed in parallel to early farmers for more than a millennium before they vanished some 4,000 years ago [7, 8]. The prolonged coexistence of the two cultures in Scandinavia has been cited as an argument against population replacement between the Mesolithic and the present [7, 8]. Through analysis of DNA extracted from ancient Scandinavian human remains, we show that people of the Pitted Ware culture were not the direct ancestors of modern Scandinavians (including the Saami people of northern Scandinavia) but are more closely related to contemporary populations of the eastern Baltic region. Our findings support hypotheses arising from archaeological analyses that propose a Neolithic or post-Neolithic population replacement in Scandinavia [7]. Furthermore, our data are consistent with the view that the eastern Baltic represents a genetic refugia for some of the European hunter-gatherer populations.

An evaluation of the genetic-matched pair study design using genome-wide SNP data from the European population.

Genetic matching potentially provides a means to alleviate the effects of incomplete Mendelian randomization in population-based gene-disease association studies. We therefore evaluated the genetic-matched pair study design on the basis of genome-wide SNP data (309,790 markers; Affymetrix GeneChip Human Mapping 500K Array) from 2457 individuals, sampled at 23 different recruitment sites across Europe. Using pair-wise identity-by-state (IBS) as a matching criterion, we tried to derive a subset of markers that would allow identification of the best overall matching (BOM) partner for a given individual, based on the IBS status for the subset alone. However, our results suggest that, by following this approach, the prediction accuracy is only notably improved by the first 20 markers selected, and increases proportionally to the marker number thereafter. Furthermore, in a considerable proportion of cases (76.0%), the BOM of a given individual, based on the complete marker set, came from a different recruitment site than the individual itself. A second marker set, specifically selected for ancestry sensitivity using singular value decomposition, performed even more poorly and was no more capable of predicting the BOM than randomly chosen subsets. This leads us to conclude that, at least in Europe, the utility of the genetic-matched pair study design depends critically on the availability of comprehensive genotype information for both cases and controls.

STR data for the AmpFlSTR Identifiler loci from Swedish population in comparison to European, as well as with non-European population.

The modern Swedish population is a mixture of people that originate from different parts of the world. This is also the truth for the clients participating in the paternity cases investigated at the department. Calculations based on a Swedish frequency database only, could give us overestimated figures of probability and power of exclusion in cases including clients with a genetic background other than Swedish. Here, we describe allele frequencies regarding the markers in the Identifiler-kit. We have compared three sets of population samples; Swedish, European and non-European to investigate how these three groups of population samples differ. Also, all three population sets were compared to data reported from other European and non-European populations. Swedish allele frequencies for the 15 autosomal STRs included in the Identifiler kit were obtained from unrelated blood donors with Swedish names. The European and non-European frequencies were based on DNA-profiles of alleged fathers from our paternity cases in 2005 and 2006.

Cryptic contamination and phylogenetic nonsense.

Ancient human DNA has been treated cautiously ever since the problems related to this type of material were exposed in the early 1990s, but as sequential genetic data from ancient specimens have been key components in several evolutionary and ecological studies, interest in ancient human DNA is on the increase again. It is especially tempting to approach archaeological and anthropological questions through this type of material, but DNA from ancient human tissue is notoriously complicated to work with due to the risk of contamination with modern human DNA. Various ways of authenticating results based on ancient human DNA have been developed to circumvent the problems. One commonly used method is to predict what the contamination is expected to look like and then test whether the ancient human DNA fulfils this prediction. If it does, the results are rejected as contamination, while if it does not, they are often considered authentic. We show here that human contamination in ancient material may well deviate from local allele frequencies or the distributions to be found among the laboratory workers and archaeologists. We conclude that it is not reliable to authenticate ancient human DNA solely by showing that it is different from what would be expected from people who have handled the material.

Analysis of linkage and linkage disequilibrium for eight X-STR markers.

X-chromosomal short tandem repeats (X-STR) have proven to be informative and useful in complex relationship testing. The main feature of X-STR markers, compared to autosomal forensic markers, is that all loci are located on the same chromosome. Thus, linkage and linkage disequilibrium may occur. The aim of this work was to study population genetic parameters of eight X-STR markers, located in four linkage groups. We present haplotype frequencies, based on 718 Swedish males, for the four linkage groups included in the Argus X-8 kit. Forensic efficiency parameters have been calculated as well as the allelic association between the tested markers for detection of linkage disequilibrium. To study the occurrences of recombination between the loci, both Swedish and Somali families were typed. A mathematical model for the estimation of recombination frequencies is presented and applied on the family samples. Our study showed that the tested markers all have highly informative forensic values and that there is a significant degree of linkage disequilibrium between the STR markers within the four linkage groups. Furthermore, based on the tested families, we also demonstrated that two of the linkage groups are partially linked. A consequence of these findings is that both linkage and linkage disequilibrium should be accounted for when producing likelihood ratios in relationship testing with X-STR markers.

Identification of mammal species using species-specific DNA pyrosequencing.

In forensic casework it is highly relevant to be able to deduce the species origin of an unknown biological sample. For such a purpose we have designed and developed an assay for species identification based on DNA sequencing of two short mitochondrial DNA amplicons. In short, partial 12S rRNA and partial 16S rRNA fragments (approximately 100bp) are amplified by PCR followed by direct sequencing using pyrosequencing technique. Due to properties of the chosen targets, the same PCR conditions and primers were used irrespective of the true species of an unknown sample. A total of 28 different mammals present in the European fauna were sequenced both for the partial 12S rRNA and the partial 16S rRNA sequences for accuracy verification. Together the two sequences showed to have a high divergence factor, discriminating almost all mammals. Furthermore, the human reference nucleotide sequences were always at least nine nucleotides different compared to the other sequenced species both at the partial 12S rRNA and the partial 16S rRNA sequences.

DNA-testing for immigration cases: the risk of erroneous conclusions.

Making the correct decision based on results from DNA analyses and other information in family reunification cases can be complicated for a number of reasons. These include stratified populations, cultural differences in family constellations, families with different population origin, and complicated family relations giving complex pedigrees. The aim of this study was to analyze the risk of erroneous conclusions in immigration cases and to propose alternative procedures to current methods to reduce the risk of making such errors. A simulation model was used to study different issues. For simplicity, we focus on cases which can be formulated as questions about paternity. We present an overview of error rates (of falsely included men as the true father and of falsely excluded true fathers) for fairly standard computations, and we show how these are affected by different factors. For example, adding more DNA markers to a case will decrease the error rates, as will the inclusion of more children. We found that using inappropriate population frequency databases had just minor effects on the error rates, but the likelihood ratios varied from an underestimation of 100 times up to an overestimation of 100,000 times. To reduce the risk of falsely including a man related to the true father we propose a more refined prior including five hypotheses instead of the two normally used. Simulations showed that this method gave reduced error rates compared with standard computations, even when the prior does not exactly correspond to reality.