Human Y Chromosome Research Articles

The fragile Y hypothesis: Y chromosome aneuploidy as a selective pressure in sex chromosome and meiotic mechanism evolution.

Loss of the Y-chromosome is a common feature of species with chromosomal sex determination. However, our understanding of why some lineages frequently lose Y-chromosomes while others do not is limited. The fragile Y hypothesis proposes that in species with chiasmatic meiosis the rate of Y-chromosome aneuploidy and the size of the recombining region have a negative correlation. The fragile Y hypothesis provides a number of novel insights not possible under traditional models. Specifically, increased rates of Y aneuploidy may impose positive selection for (i) gene movement off the Y; (ii) translocations and fusions which expand the recombining region; and (iii) alternative meiotic segregation mechanisms (achiasmatic or asynaptic). These insights as well as existing evidence for the frequency of Y-chromosome aneuploidy raise doubt about the prospects for long-term retention of the human Y-chromosome despite recent evidence for stable gene content in older non-recombining regions.

Gene pool similarities and differences between Ukrainians and Russians of Slobozhanshchina based on Y-chromosome data

The results of the study of Y-chromosomal polymorphisms of Russian and Ukrainian population are presented for Slobozhanshina--contemporary border region, former "Wild Field" boundary, which was inhabited in XVII-XVIII centuries by both the Russians from the north and Ukrainians from the west. In general, Ukrainian and Russian populations of Slobozhanshchina genetically are very close, their set and frequency range of Y-chromosome haplogroups are typical for the Eastern Europe. But a detailed analysis of highly informative Y-chromosome markers showed that after 3,5 centuries of coexistence on the same historical territory, the both nations retain the ethnic specificity of their gene pools: Ukrainian populations are similar to the rest of Ukraine, and Russian populations are similar to the south of the European part of Russia. The genetic differences may be due to the spatial characteristics of marriage migration and the predominant ethnic environment.

Vestiges of an Ancient Border in the Contemporary Genetic Diversity of North-Eastern Europe.

It has previously been demonstrated that the advance of the Neolithic Revolution from the Near East through Europe was decelerated in the northernmost confines of the continent, possibly as a result of space and resource competition with lingering Mesolithic populations. Finland was among the last domains to adopt a farming lifestyle, and is characterized by substructuring in the form of a distinct genetic border dividing the northeastern and southwestern regions of the country. To explore the origins of this divergence, the geographical patterns of mitochondrial and Y-chromosomal haplogroups of Neolithic and Mesolithic ancestry were assessed in Finnish populations. The distribution of these uniparental markers revealed a northeastern bias for hunter-gatherer haplogroups, while haplogroups associated with the farming lifestyle clustered in the southwest. In addition, a correlation could be observed between more ancient mitochondrial haplogroup age and eastern concentration. These results coupled with prior archeological evidence suggest the genetic northeast/southwest division observed in contemporary Finland represents an ancient vestigial border between Mesolithic and Neolithic populations undetectable in most other regions of Europe.

Common AZFc structure may possess the optimal spermatogenesis efficiency relative to the rearranged structures mediated by non-allele homologous recombination.

The azoopsermia factor c (AZFc) region of human Y-chromosome is an essential genomic segment for spermatogenesis with frequent non-allele homologous recombination (NAHR). Recent case-control studies on the association of the NAHR-based AZFc structural mutations with spermatogenic failure produced inconsistent results. To more precisely evaluate their spermatogenesis effects, we investigated the correlation between the subdivided AZFc mutations and sperm production in 3,439 Han Chinese males. Our results showed that both partial AZFc deletion-only and primary duplication mutation presented a significant risk for decreased sperm production. Restoration of the reduced dosage of the AZFc content to the normal level had a milder effect, whereas an overdose of the AZFc content arising from multiple duplications of a partial AZFc-deleted structure produced a more serious consequence compared to the partial deletion-only mutation. Additionally, the AZFc-mutated structures with excessive NAHR-substrate showed a notably negative effect on spermatogenesis. These results suggest that the recurrent NAHR-based AZFc mutations may be associated with decreased spermatogenesis efficiency in present population. More significantly, our finding implies that the overdose of AZFc NAHR-substrate may produce an additional risk for the massive AZFbc deletions during the multi-stage division process of germ cells and thus impair the global spermatogenesis efficiency in the carriers.

Genome-wide signatures of male-mediated migration shaping the Indian gene pool.

Multiple questions relating to contributions of cultural and demographical factors in the process of human geographical dispersal remain largely unanswered. India, a land of early human settlement and the resulting diversity is a good place to look for some of the answers. In this study, we explored the genetic structure of India using a diverse panel of 78 males genotyped using the GenoChip. Their genome-wide single-nucleotide polymorphism (SNP) diversity was examined in the context of various covariates that influence Indian gene pool. Admixture analysis of genome-wide SNP data showed high proportion of the Southwest Asian component in all of the Indian samples. Hierarchical clustering based on admixture proportions revealed seven distinct clusters correlating to geographical and linguistic affiliations. Convex hull overlay of Y-chromosomal haplogroups on the genome-wide SNP principal component analysis brought out distinct non-overlapping polygons of F*-M89, H*-M69, L1-M27, O2a-M95 and O3a3c1-M117, suggesting a male-mediated migration and expansion of the Indian gene pool. Lack of similar correlation with mitochondrial DNA clades indicated a shared genetic ancestry of females. We suggest that ancient male-mediated migratory events and settlement in various regional niches led to the present day scenario and peopling of India.

Surnames and Y-chromosomal markers reveal low relationships in Southern Spain.

A sample of 416 males from western and eastern Andalusia has been jointly analyzed for surnames and Y-chromosome haplogroups and haplotypes. The observed number of different surnames was 222 (353 when the second surname of the Spanish system of naming is considered). The great majority of recorded surnames have a Castilian-Leonese origin, while Catalan or Basque surnames have not been found. A few Arab-related surnames appear but none discernible of Sephardic-Jewish descent. Low correlation among surnames with different population frequencies and Y-chromosome markers, at different levels of genetic resolution, has been observed in Andalusia. This finding could be explained mainly by the very low rate of monophyletic surnames because of the historical process of surname ascription and the resulting high frequencies of the most common Spanish surnames. The introduction of surnames in Spain during the Middle Ages coincided with Reconquest of the territories under Islamic rule, and Muslims and Jews progressively adopted the present male line surname system. Sampled surnames and Y-chromosome lineages fit well a power-law distribution and observed isonymy is very close to that of the general population. Besides, our data and results show that the reliability of the isonymy method should be questioned because of the high rate of polyphyletic surnames, even in small geographic regions and autochthonous populations. Random isonymy would be consistently dependent of the most common surname frequencies in the population.

Deep phylogenetic analysis of haplogroup G1 provides estimates of SNP and STR mutation rates on the human Y-chromosome and reveals migrations of Iranic speakers.

Y-chromosomal haplogroup G1 is a minor component of the overall gene pool of South-West and Central Asia but reaches up to 80% frequency in some populations scattered within this area. We have genotyped the G1-defining marker M285 in 27 Eurasian populations (n= 5,346), analyzed 367 M285-positive samples using 17 Y-STRs, and sequenced ~11 Mb of the Y-chromosome in 20 of these samples to an average coverage of 67X. This allowed detailed phylogenetic reconstruction. We identified five branches, all with high geographical specificity: G1-L1323 in Kazakhs, the closely related G1-GG1 in Mongols, G1-GG265 in Armenians and its distant brother clade G1-GG162 in Bashkirs, and G1-GG362 in West Indians. The haplotype diversity, which decreased from West Iran to Central Asia, allows us to hypothesize that this rare haplogroup could have been carried by the expansion of Iranic speakers northwards to the Eurasian steppe and via founder effects became a predominant genetic component of some populations, including the Argyn tribe of the Kazakhs. The remarkable agreement between genetic and genealogical trees of Argyns allowed us to calibrate the molecular clock using a historical date (1405 AD) of the most recent common genealogical ancestor. The mutation rate for Y-chromosomal sequence data obtained was 0.78×10-9 per bp per year, falling within the range of published rates. The mutation rate for Y-chromosomal STRs was 0.0022 per locus per generation, very close to the so-called genealogical rate. The “clan-based” approach to estimating the mutation rate provides a third, middle way between direct farther-to-son comparisons and using archeologically known migrations, whose dates are subject to revision and of uncertain relationship to genetic events.

Deletion and duplication at DYS448 and DYS626 loci: unexpected patterns within the AZFc region of the Y-chromosome.

Increasing the knowledge of multiple and microstructural events within the Y-chromosome may prove useful to better characterize abnormal short tandem repeats patterns complicating DNA profile interpretation. On the long arm of the human Y-chromosome, such structural rearrangements were observed in azoospermia factor regions (AZFa, AZFb, AZFc) that play an important role in male fertility and also host Y-STRs commonly used in forensic genetics. Here, we describe two cases, involving two males formerly included in an Italian population study, where DYS448 and DYS626 loci, located within the AZFc region, simultaneously displayed a double deletion in one case and a double duplication in the other. With the aim of better defining the size of both events, low and high-resolution mapping by means of 16 sequence-tagged sites was performed, and unexpected discontinued patterns within the palindromic segments b1/b3 of the AZFc were identified. Extending the analysis to their respective male relatives revealed unaltered transmission of the patterns along the two pedigrees. Reviewing literature data describing DYS448-DYS626 deletion and duplication suggested no close correlation between the occurrence of multiple/microstructural events and geographical origin.

Paternal lineages in Libya inferred from Y-chromosome haplogroups.

Many studies based on genetic diversity of North African populations have contributed to elucidate the modelling of the genetic landscape in this region. North Africa is considered as a distinct spatial-temporal entity on geographic, archaeological, and historical grounds, which has undergone the influence of different human migrations along its shaping. For instance, Libya, a North African country, was first inhabited by Berbers and then colonized by a variety of ethnic groups like Phoenicians, Greeks, Romans, Arabs and, in recent times, Italians. In this study, we contribute to clarify the genetic variation of Libya and consequently, of North African modern populations, by the study of Libyan male lineages. A total of 22 Y-chromosome-specific SNPs were genotyped in a sample of 175 Libyan males, allowing the characterization of 18 Y-chromosomal haplogroups. The obtained data revealed a predominant Northwest African component represented by haplogroup E-M81 (33.7%) followed by J(xJ1a,J2)-M304 (27.4%), which is postulated to have a Middle Eastern origin. The comparative study with other populations (∼5,400 individuals from North Africa, Middle East, Sub-Saharan Africa, and Europe) revealed a general genetic homogeneity among North African populations (FST = 5.3 %; P-value < 0.0001). Overall, the Y-haplogroup diversity in Libya and in North Africa is characterized by two genetic components. The first signature is typical of Berber-speaking people (E-M81), the autochthonous inhabitants, whereas the second is (J(xJ1a,J2)-M304), originating from Arabic populations. This is in agreement with the hypothesis of an Arabic expansion from the Middle East, shaping the North African genetic landscape.

Distribution of East Eurasian Y-Chromosome and Mitochondrial DNA Haplogroups across Eurasia: Insights into the Genetic Ancestry of Bulgarians

The modern Bulgarian mitochondrial DNA (mtDNA) and Y-chromosome gene pools predominantly consist of Western Eurasian haplogroups. In contrast, the Eastern Eurasian lineages are found at very low frequencies in Bulgarians, being represented only by mtDNA haplogroups C (0.2%), D (0.4%) and Z (0.1%) (Karachanak et al., 2012) and Y-chromosome haplogroups C, N and Q (each 0.5%) (Karachanak et al., 2013). A similar pattern is observed in ancient mtDNA samples of proto-Bulgarian human remains, which belong exclusively to Western Eurasian mtDNA haplogroups (Nesheva et al., 2015). In order to investigate Bulgarian ancestry from the perspective of Eastern Eurasian haplogroups, we have analyzed the distribution of Y-chromosome haplogroups C, N and Q and mtDNA haplogroups C, D and Z across Eurasia. The survey was performed using literature data for more than 15,000 individuals from different Eurasian (sub-) populations for each of these haplogroups. The collected data were used to construct Eurasian frequency maps of the considered haplogroups and to test the significance of their incidence between Bulgarians and Europeans, European neighboring populations of Bulgaria and populations, which according to some historical conceptions could have common ancestry with proto-Bulgarians, namely: Altaian, Caucasus, Siberian and Central Asian populations. The spatial distribution of mtDNA haplogroups C, D and Z and Y-chromosome haplogroups C, N and Q contrasts their high frequency among Altaic populations and their occasional appearance in Bulgarians. Furthermore, the comparison of the occurrence of these haplogroups shows no link between Bulgarians and Altaic and Caucasus populations. Based on the substantial genetic input of proto-Bulgarians to the modern Bulgarian gene pool, the present study confirms the nonexistence of a close Y-chromosomal or mtDNA link between proto-Bulgarians on the one hand and Altaic and Caucasus populations on the other.

Genetic characterization of populations of the Volga-Ural region according to the variability of the Y-chromosome

An analysis of genetic diversity in nine ethnic groups of the Volga-Ural region was carried out using 15 biallelic loci in the nonrecombining region of the Y-chromosome. The major Y-chromosome haplogroups in the region are R1a-M198, R1b-M269, and N-M231. It was found that Bashkirs show the greatest difference from other populations of the Volga-Ural region according both to F(st) and to the principal component analysis. In addition, analysis of the frequency distribution of Y-chromosome haplogroups was carried out in the Besermyan population, which was not studied previously from the Y-chromosome perspective. The results of this study revealed the predominance of haplogroup N-M231 (54.7%) in this ethnic group, which may indicate the prevalence of the Finno-Ugric component in the formation of the patrilineal component in the gene pool of the Besermyan ethnic group.

The Major Y-Chromosome Haplogroup R1b-M269 in West-Europe, Subdivided by the Three SNPs S21/U106, S145/L21 and S28/U152, Shows a Clear Pattern of Geographic Differentiation

More than 2600 unrelated males from West-Europe were analysed by molecular hybridization experiments for the p49a,fTaq I polymorphisms. A total of 895 subjects (34%), belonging to haplogroup M269, were identified and further analysed for the three SNPs, S21/U106, S145/L21 and S28/U152; these three SNPs define the Northwest, West and South European sub-haplogroups, respectively. These haplogroups showed quite different frequency distribution patterns within West-Europe, with frequency peaks in Northern Europe, in Brittany in France and in Northern Italy/Southern France.

Complex Analysis of 700-Year-Old Skeletal Remains found in an Unusual Grave–Case Report

Aim: The present study was designed to analyze the 700-year-old human remains from an unusual grave using a combined approach that consisted of anthropological, archaeogenetic, genealogical, mass spectrometry, 3-dimensional (3D) modeling and facial reconstruction methods to confirm or reject several hypotheses about the skeletal remains. Methods: DNA was extracted from the skeleton and amplified using autosomal and Y-chromosome human identification short tandem repeat (STR) kits that were designed for forensic use, and sequence data were obtained from hyper variable region I (HVRI) mtDNA sequencing. Elemental mapping and quantification of investigated elements were performed using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The Computed Tomography (CT) images of the skull were created in a transversal plane, and the scans were used to create 3D geometric models of the skull. A plastic physical model (a cast) of the skull was produced by rapid prototyping technology, and the model was used for sculptural facial approximation of the studied individual. Results: The Y-chromosome haplogroup of the sample was determined to be E1b1b, and the assigned mtDNA haplogroup was H. LA-ICP-MS and geochemical analysis revealed that the individual consumed plants and meats, except pork. Anthropological examination estimated the age of the individual to be between 45-55 years, and we did not find any traces of disablement or physical anomalies. Interestingly, we were able to produce a facial reconstruction according to the skull. Conclusion: Applying a multidisciplinary approach to the examination of the 14th century material enabled us to retrieve new types of information that helped us to interpret the excavated skeletal remains.

Diversity of killer cell immunoglobulin-like receptor genes in the Bengali population of northern West Bengal, India.

The Indian Subcontinent exhibits extensive diversity in its culture, religion, ethnicity and linguistic heritage, which symbolizes extensive genetic variations within the populations. The highly polymorphic Killer cell Immunoglobulin-like Receptor (KIR) family plays an important role in tracing genetic differentiation in human population. In this study, we aimed to analyse the KIR gene polymorphism in the Bengali population of northern West Bengal, India. To our knowledge, this is the first report on the KIR gene polymorphism in the Bengalis of West Bengal, India. Herein, we have studied the distribution of 14 KIR genes (KIR3DL1-3DL3, KIR2DL1-2DL5, KIR2DS1-2DS5 AND KIR3DS1) and two pseudogenes (KIR3DP1 and 2DP1) in the Bengalis. Apart from the framework genes (KIR2DL4, 3DL2, 3DL3 and 3DP1), which are present in all the individuals, the gene frequencies of other KIR genes varied between 0.34 and 0.88. Moreover, upon comparing the KIR polymorphism of the Bengalis with the available published data of other world populations, it has been found that the Indo-European-speaking Bengalis from the region share both Dravidian and Indo-Aryan gene pool with considerable influences of mongoloid and European descents. Furthermore, evidences from previously published data on human leucocyte antigen and Y-chromosome haplogroup diversity support the view. Our results will help to understand the genetic background of the Bengali population, in illustrating the population migration events in the eastern and north-eastern part of India, in explaining the extensive genetic admixture amongst the different linguistic groups of the region and also in KIR-related disease researches.

Towards a consensus Y-chromosomal phylogeny and Y-SNP set in forensics in the next-generation sequencing era

Currently, several different Y-chromosomal phylogenies and haplogroup nomenclatures are presented in scientific literature and at conferences demonstrating the present diversity in Y-chromosomal phylogenetic trees and Y-SNP sets used within forensic and anthropological research. This situation can be ascribed to the exponential growth of the number of Y-SNPs discovered due to mostly next-generation sequencing (NGS) studies. As Y-SNPs and their respective phylogenetic positions are important in forensics, such as for male lineage characterization and paternal bio-geographic ancestry inference, there is a need for forensic geneticists to know how to deal with these newly identified Y-SNPs and phylogenies, especially since these phylogenies are often created with other aims than to carry out forensic genetic research. Therefore, we give here an overview of four categories of currently used Y-chromosomal phylogenies and the associated Y-SNP sets in scientific research in the current NGS era. We compare these categories based on the construction method, their advantages and disadvantages, the disciplines wherein the phylogenetic tree can be used, and their specific relevance for forensic geneticists. Based on this overview, it is clear that an up-to-date reduced tree with a consensus Y-SNP set and a stable nomenclature will be the most appropriate reference resource for forensic research. Initiatives to reach such an international consensus are therefore highly recommended.

Y-SNP L1034: limited genetic link between Mansi and Hungarian-speaking populations.

Genetic studies noted that the Hungarian Y-chromosomal gene pool significantly differs from other Uralic-speaking populations. Hungarians show very limited or no presence of haplogroup N-Tat, which is frequent among other Uralic-speaking populations. We proposed that some genetic links need to be observed between the linguistically related Hungarian and Mansi populations.This is the first attempt to divide haplogroup N-Tat into subhaplogroups by testing new downstream SNP markers L708 and L1034. Sixty Northern Mansi samples were collected in Western Siberia and genotyped for Y-chromosomal haplotypes and haplogroups. We found 14 Mansi and 92 N-Tat samples from 7 populations. Comparative results showed that all N-Tat samples carried the N-L708 mutation. Some Hungarian, Sekler, and Uzbek samples were L1034 SNP positive, while all Mongolians, Buryats, Khanty, Finnish, and Roma samples yielded a negative result for this marker. Based on the above, L1034 marker seems to be a subgroup of N-Tat, which is typical for Mansi and Hungarian-speaking ethnic groups so far. Based on our time to most recent common ancestor data, the L1034 marker arose 2,500 years before present. The overall frequency of the L1034 is very low among the analyzed populations, thus it does not necessarily mean that proto-Hungarians and Mansi descend from common ancestors. It does provide, however, a limited genetic link supporting language contact. Both Hungarians and Mansi have much more complex genetic population history than the traditional tree-based linguistic model would suggest.

Biochip for genotyping SNPs defining core Y-chromosome haplogroups in Russian population groups

A biochip Ycore10 has been developed which allows identification of the following SNP markers of Y haplogroups: M130 (C), M145 (DE), P257 (G), M69(H), U179 (I), M304 (J), M185 (L), M231 (N), M175 (O) and P224 (R). The biochip can be used in determination of paternal ethnicity of offenders in the complex ethnic milieu of Russia. The sensitivity limit of the biochip-based assay was 31 pg of DNA template. Ycore10 genotyping was successfully performed with a range of male DNA concentrations (0.5–300 ng). Altogether 89 samples were genotyped and the following haplogroups were detected: C (2), DE (10), G (6), H (2), I (20), J (6), L (1), N (17), O (2), R (22). In one sample the ancestral alleles were detected for all ten biallelic markers. This tool is feasible for genotyping of mixed and degraded samples.

Y-chromosome analysis in a Northwest Iberian population: unraveling the impact of Northern African lineages.

To provide new clues about the genetic origin, composition and structure of the population of the Spanish province of Zamora, with an emphasis on the genetic impact of the period of Islamic rule in the Iberian Peninsula. Polymorphisms in the paternally inherited Y-chromosome, Single Nucleotide Polymorphisms and Short Tandem Repeats, were analyzed in 235 unrelated males born in six different regions in the Zamora province. A relatively homogenous Y-chromosome haplogroup composition was observed in the Zamora province. Haplogroups R1b1-P25 and I-M170, widespread in European populations, accounted for 64.9% of the total sample. Moreover, all of the observed African lineages, accounting for 10.2% of the total variability, belonged to haplogroups having Northwest African origin (E1b1b1b-M81, E1b1b1a-β-M78, and J1-M267). No differences between regions or sub-structure due to geographical boundaries were detected. The specific Northwest African male lineages observed contrast with the mitochondrial DNA data, where the majority of African lineages were found to be sub-Saharan. This work made it possible to study the impact of recent historical events in the male gene pool in the province of Zamora in Spain.

An updated phylogeny of the human Y-chromosome lineage O2a-M95 with novel SNPs.

Though the Y-chromosome O2a-M95 lineage is one of the major haplogroups present in eastern Asian populations, especially among Austro-Asiatic speaking populations from Southwestern China and mainland Southeast Asia, to date its phylogeny lacks structure due to only one downstream SNP marker (M88) assigned to the lineage. A recent array-capture-based Y chromosome sequencing of Asian samples has yielded a variety of novel SNPs purportedly belonging to the O2a-M95 lineage, but their phylogenetic positions have yet to be determined. In this study, we sampled 646 unrelated males from 22 Austro-Asiatic speaking populations from Cambodia, Thailand and Southwestern China, and genotyped 12 SNP makers among the sampled populations, including 10 of the newly reported markers. Among the 646 males, 343 belonged to the O2a-M95 lineage, confirming the supposed dominance of this Y chromosome lineage in Austro-Asiatic speaking populations. We further characterized the phylogeny of O2a-M95 by defining 5 sub-branches: O2a1*-M95, O2a1a-F789, O2a1b*-F1252, O2a1b1*-M88 and O2a1b1a -F761. This updated phylogeny not only improves the resolution of this lineage, but also allows for greater tracing of the prehistory of human populations in eastern Asia and the Pacific, which may yield novel insights into the patterns of language diversification and population movement in these regions.

Evaluation of the Ion Torrent™ HID SNP 169-plex: A SNP typing assay developed for human identification by second generation sequencing.

The Ion Torrent™ HID SNP assay amplified 136 autosomal SNPs and 33 Y-chromosome markers in one PCR and the markers were subsequently typed using the Ion PGM™ second generation sequencing platform. A total of 51 of the autosomal SNPs were selected from the SNPforID panel that is routinely used in our ISO 17025 accredited laboratory. Concordance between the Ion Torrent™ HID SNP assay and the SNPforID assay was tested by typing 44 Iraqis twice with the Ion Torrent™ HID SNP assay. The same samples were previously typed with the SNPforID assay and the Y-chromosome haplogroups of the individuals were previously identified by typing 45 Y-chromosome SNPs. Full concordance between the assays were obtained except for the SNP genotypes of two SNPs. These SNPs were among the eight SNPs (rs2399332, rs1029047, rs10776839, rs4530059, rs8037429, rs430046, rs1031825 and rs1523537) with inconsistent allele balance among samples. These SNPs should be excluded from the panel. The optimal amount of DNA in the PCR seemed to be ≥0.5ng. Allele drop-outs were rare and only seen in experiments with <0.5ng input DNA and with a coverage of <50reads. No allele drop-in was observed. The great majority of the heterozygote allele balances were between 0.6 and 1.6, which is comparable to the heterozygote balances of STRs typed with PCR-CE. The number of reads with base calls that differed from the genotype call was typically less than five. This allowed detection of 1:100 mixtures with a high degree of certainty in experiments with a high total depth of coverage. In conclusion, the Ion PGM™ is a very promising platform for forensic genetics. However, the secondary sequence analysis software made wrong genotype calls from correctly sequenced alleles. These types of errors must be corrected before the platform can be used in case work. Furthermore, the sequence analysis software should be further developed and include quality settings for each SNP based on validation studies.