Y-chromosome Markers Research Articles

Paleoasian Substrate in the Gene Pool of Koryaks According to Data on Autosomal SNP Polymorphism and Y-Chromosome Haplogroups

The gene pool of the Koryaks was studied in comparison with other Far Eastern and Siberian peoples using a genome-wide panel of autosomal single-nucleotide polymorphic markers and Y-chromosome markers. The results of analyzing the frequencies of autosomal SNPs using various methods, the similarity in the composition of Y-chromosome haplogroups and YSTR haplotypes indicate that the gene pool of the Koryaks is as close as possible to the Chukchi one and was formed as a result of the unification of several groups whose ancestors moved from the territory of modern Yakutia and the Amur region. The two dominant Y-chromosome haplogroups of the Koryaks with different sublineages and haplotype clusters demonstrate their contacts with the Chukchi, Evens, Yukaghirs and Eskimos. Analysis of the composition of genetic components and IBD blocks on autosomes indicates the maximum genetic proximity of the Koryaks to the Chukchi. Among the Siberian populations, the Chukchi, Koryaks and Nivkhs form a separate cluster from the main group of Siberian populations, while the Chukchi and Koryaks are more closely related. Far Eastern populations are divided in full accordance with geographic localization into the northern group (Chukchi and Koryaks) and the southern group, including the Nivkhs and Udege. A more detailed analysis of the component composition of gene pools in some populations reveals components specific to them. The isolation of such components is associated with founder effects and a shift in allele frequencies for these populations. The Koryaks and Chukchi are one of the most striking examples of long-standing genetic kinship. In their populations, maximum values of the level of genomic inbreeding FROH 1.5 (0.0422, 0.0409) were found, which is natural due to their relative isolation.

Генетические портреты хантов и манси по гаплогруппам Y-хромосомы в контексте генофондов России

Khanty and Mansi are small indigenous peoples of Western Siberia with the unique cultural, anthropological, and linguistic characteristics. The study of their gene pool will make it possible to reconstruct the genetic structure of the Ugric-speaking population, of which in modern times only Khanty and Mansi remain, along with Hungarians, in whose gene pool there are traces of medieval migration of the Ugric-speaking Magyars. The detailed characterization of the gene pool of Khanty and Mansi is important for reconstruction of Ugric populations and genetic history of the region. The study was aimed to assess representative samples of Khants (n = 83) and Mansi (n = 74) based on the standard panel of 60 SNP markers and the extended panel of 74 Y-chromosomal SNP markers by statistical and cartographic methods in the context of indigenous population of Urals and Western Siberia. The differences between the gene pools of Khanty and Mansi have been revealed based on both standard panel of Y chromosome haplogroups and branches of haplogroups N2 and N3a4. Most of the Khanty gene pool is evenly distributed between N2-Y3195 (26%), N2-VL67 (23%), and N3a4-Z1936 (23%). The “Western” branch N2-Y3195 predominates in the Mansi gene pool (69%). Mansi gravitate towards populations of the Urals-Volga region in the multidimensional genetic space. Based on the standard panel of Y haplogroups, Khanty are close to the populations of Western and South Siberia. However, the analysis of branches N3a4 has shown that Khanty are intermediate between the “Uralic” and “Siberian” clusters: when the ancestors of Khanty moved from the Ural region to the northeast, these acquired both genetic components. The gene geographic maps of 10 haplogroup N3a4 branches in the populations of Urals and Western Siberia reflect the dynamic changes of the gene pool that took place 4–2 kya.

Expanding the scope of methylation-sensitive restriction enzyme (MSRE) PCR for forensic identification of body fluids through the novel use of methylation-dependent restriction enzymes (MDRE) and the combination of autosomal and Y-chromosomal markers

Methylation-sensitive/-dependent restriction enzyme (MSRE/MDRE) PCR can be performed to detect hypomethylated or hypermethylated CpG sites. With the combined use of different tissue-specific CpG markers, MSRE/MDRE-PCR leads to tissue-specific methylation patterns (TSMPs), enabling the correlation of DNA samples to their source tissue. MSRE/MDRE assays can use the same platform as forensic STR typing and offer many advantages in the field of forensic body fluid detection. In the present study, we aimed to establish MSRE assays for the detection of blood, saliva, vaginal secretion, and semen, using markers from literature and from our own database search. We designed two different MSRE test-sets, which include two novel Y-chromosomal non-semen markers, and enable differentiation between female and male non-semen samples. Furthermore, we established an MSRE/MDRE semen approach, which includes only Y-chromosomal non-semen and semen markers. This Y-semen multiplex PCR utilizes the novel combination of the methylation-sensitive enzyme SmaI and the methylation-dependent enzyme GlaI, which enables more sensitive detection of male body fluids within male/female DNA mixtures. Our validation tests confirmed that MSRE/MDRE assays exhibit high sensitivity, similar to that of STR typing.

Validation of phylogenetic informative Y-InDels in Y-chromosomal haplogroup O-M175.

The Y-chromosomal haplogroup tree, which consists of a group of Y-chromosomal loci with phylogenetic information, has been widely applied in anthropology, archaeology and population genetics. With the continuous updating of the phylogenetic structure, Y-chromosomal haplogroup tree provides more information for recalling the biogeographical origin of Y chromosomes. Generally, Y-chromosomal insertion-deletion polymorphisms (Y-InDels) are genetically stable as Y-chromosomal single nucleotide polymorphisms (Y-SNPs), and therefore carry mutations that can accumulate over generations. In this study, potential phylogenetic informative Y-InDels were filtered out in haplogroup O-M175, which is dominant in East Asia, based on population data retrieved from the 1000 Genomes Project. A group of 22 phylogenetic informative Y-InDels were identified and then assigned to their corresponding subclades of haplogroup O-M175, which provided a supplement for the update and application of Y-chromosomal markers. Especially, four Y-InDels were introduced to define subclades determined using a single Y-SNP.

Relationship of the gene pool of the Khants with the peoples of Western Siberia, Cis-Urals and the Altai-Sayan Region according to the data on the polymorphism of autosomic locus and the Y-chromosome.

Khanty are indigenous Siberian people living on the territory of Western Siberia, mainly on the territory of the Khanty-Mansiysk and Yamalo-Nenets Autonomous Okrugs. The present study is aimed at a comprehensive analysis of the structure of the Khanty gene pool and their comparison with other populations of the indigenous population of Southern and Western Siberia. To address the issues of genetic proximity of the Khanty with other indigenous peoples, we performed genotyping of a wide genomic set of autosomal markers using high-density biochips, as well as an expanded set of SNP and STR markers of the Y-chromosome in various ethnic groups: Khakas, Tuvans, Southern Altaians, Siberian Tatars, Chulyms (Turkic language family) and Kets (Yeniseian language family). The structure of the gene pool of the Khanty and other West Siberian and South Siberian populations was studied using a genome-wide panel of autosomal single nucleotide polymorphic markers and Y-chromosome markers. The results of the analysis of autosomal SNPs frequencies by various methods, the similarities in the composition of the Y-chromosome haplogroups and YSTR haplotypes indicate that the Khanty gene pool is quite specific. When analyzing autosomal SNPs, the Ugrian genetic component completely dominates in both samples (up to 99-100 %). The samples of the Khanty showed the maximum match in IBD blocks with each other, with a sample of the Kets, Chulyms, Tuvans, Tomsk Tatars, Khakas, Kachins, and Southern Altaians. The degree of coincidence of IBD blocks between the Khanty, Kets, and Tomsk Tatars is consistent with the results of the distribution of allele frequencies and common genetic components in these populations. According to the composition of the Y-chromosome haplogroups, the two samples of the Khanty differ significantly from each other. A detailed phylogenetic analysis of various Y-chromosome haplogroups made it possible to describe and clarify the differences in the phylogeny and structure of individual ethnospecific sublines, to determine their relationship, traces of population expansion in the Khanty gene pool. Variants of different haplogroups of the Y-chromosome in the Khanty, Khakas and Tuvans go back to their common ancestral lines. The results of a comparative analysis of male samples indicate a close genetic relationship between the Khanty and Nenets, Komi, Udmurts and Kets. The specificity of haplotypes, the discovery of various terminal SNPs confirms that the Khanty did not come into contact with other ethnic groups for a long time, except for the Nenets, which included many Khanty clans.

Structure and origin of Tuvan gene pool according to autosome SNP and Y-chromosome haplogroups.

Tuvans are one of the most compactly living peoples of Southern Siberia, settled mainly in the territory of Tuva. The gene pool of the Tuvans is quite isolated, due to endogamy and a very low frequency of interethnic marriages. The structure of the gene pool of the Tuvans and other Siberian populations was studied using a genome-wide panel of autosomal single nucleotide polymorphic markers and Y-chromosome markers. The results of the analysis of the frequencies of autosomal SNPs by various methods, the similarities in the composition of the Y-chromosome haplogroups and YSTR haplotypes show that the gene pool of the Tuvans is very heterogeneous in terms of the composition of genetic components. It includes the ancient autochthonous Yeniseian component, which dominates among the Chulym Turks and Kets, the East Siberian component, which prevails among the Yakuts and Evenks, and the Far Eastern component, the frequency of which is maximum among the Nivkhs and Udeges. Analysis of the composition of IBD-blocks on autosomes shows the maximum genetic relationship of the Tuvans with the Southern Altaians, Khakas and Shors, who were formed during the settlement of the Turkic groups of populations on the territory of the Altai-Sayan region. A very diverse composition of the Tuvan gene pool is shown for various sublines of Y-chromosomal haplogroups, most of which show strong ethnic specificity. Phylogenetic analysis of individual Y-chromosome haplogroups demonstrates the maximum proximity of the gene pool of the Tuvans with the Altaians, Khakas and Shors. Differences in frequencies of Y-chromosome haplogroups between the Todzhans and Tuvans and a change in the frequencies of haplogroups from south to north associated with the East Asian component were found. The majority of the most frequent Y-chromosome haplogroups in the Tuvans demonstrate the founder effect, the formation age of which is fully consistent with the data on their ethnogenesis.

Genetic differentiation of Andaman Islanders and their relatedness to Nicobar Islanders

The Andaman Islanders, living in isolation for centuries, have been subject to recent molecular scrutiny with their Asian origins being established. Although marked linguistic and physical variations are known to exist among the different extant population groups, the underlying genetic structure with reference to indigenous populations of adjacent Nicobar Islands has been poorly understood. To decipher the genetic structure of three indigenous Andaman Islanders; the Great Andamanese, Jarawa and the Onge, molecular analyses with autosomal, mitochondrial and Y-chromosomal markers were carried out and compared with indigenous Nicobar Islanders; the Nicobarese and Shompen.Biparental analyses revealed the Great Andamanese exhibiting considerable population substructuring owing to their present-day conglomeration of several erstwhile segregate tribes, in addition to discernable admixture from mainland Indian populations and other Andaman and Nicobar Islanders especially the Nicobarese. Distant separation between the Great Andamanese and Nicobarese was also discerned by the shared R22 mitochondrial lineage. The R22 lineage was previously reported in the Shompen of Nicobar Islands and Southeast Asian populations. The Nicobarese also exhibited mitochondrial haplogroup M74b, which has a Southeast Asian and East Asian distribution.The Onge-Jarawa group demonstrated a close genetic relatedness, consistent with their linguistic proximity. However, recent differentiation of the Jarawa from the Onge was also inferred from their separation on both mitochondrial M31 and M32 lineages as well as from autosomal and Y-chromosomal divergences. Our analyses indicate considerable genetic drift among the Andaman Islanders, a result of long-term isolation and inbreeding.Autosomal and mitochondrial data suggest considerable admixture between the ‘Negrito’ Great Andamanese and ‘non-Negrito’ Nicobarese.

DETECTION OF Y-CHROMOSOME MARKER IN PLASMA OF PREGNANT WOMEN USING REAL TIME PCR: DIAGNOSTIC ACCURACY DEPENDING ON GESTATION AGE

The evaluation of the clinical significance of the test for the detection of the Y-chromosome marker in the plasma of a pregnant woman at different stages of pregnancy by real-time PCR was carried out. The blood samples of 4616 women at 4 to 32 gestation weeks were studied. Identification of the Y-chromosome marker was carried out based on the amplification of a region of the TSPY gene. The Y-chromosome marker was unambiguously identified in 2131 samples, which accounted for 46.2% of the total number of analyzed samples. In 233 samples (5%), the Y-chromosome marker was detected with reduced reliability, and in 15 samples (0.3%), an unambiguous conclusion about the presence or absence of Y-specific DNA in plasma could not be made during the initial study. The diagnostic accuracy of the Y-chromosome marker determination in the plasma of a pregnant woman at the 4-6th gestation week was 95.5%, and from the 7th week and at later stages of pregnancy it reached 97.3-98.2%. Testing from the 7th gestation week may be recommended for reliable prenatal sex determination of the fetus by real-time PCR analysis of extracellular circulating fetal DNA.

Y-chromosome diversity in dairy bulls of Tamil Nadu

The Y-chromosome specific microsatellite studies are of particular interest as they could reveal the paternal lineage and domestication pattern among the population distributed over geographical area. As few bulls are used against large number of cows using artificial insemination approach, it is necessary to study the Y-chromosome microsatellite markers to understand the Y-chromosome diversity, paternal lineage and molecular variation among population. The present study included 431 bulls comprising Bos taurus, Bos indicus and crossbreds from various frozen semen stations of Tamil Nadu. The microsatellites loci viz.UMN0103, UMN0307, BM861, UMN0504, INRA189, INRA124,DDX3Y and UMN2008 were screened. The overall mean number of different alleles, number of effective alleles, Shannon’s information index, diversity and unbiased diversity were 2.18 ±0.16, 1.37 ± 0.06, 0.35 ± 0.04, 0.20 ± 0.02 and 0.21 ± 0.02 respectively. The analysis of molecular variance revealed that the variation within populations accounted for 66 per cent of total variation.

Türk İnfertil Erkeklerde Y kromozomu Mikrodelesyonlarının Sıklığı: Tek Merkez Deneyimi

Objective: Infertility is defined as the failure to achieve a clinical pregnancy after twelve months of regular unprotected sexual intercourse. Both genetic and environmental factors affect infertility. The aim of the study is to establish the frequency of the Y chromosome microdeletions in Turkish infertile men who were referred to our center (2016-2020) with severe oligozoospermia and azoospermia.
 Methods: A retrospective chart review study on patients who referred to our center between 2016-2020 due to infertility were included in the study. We evaluated microdeletions of the Y-chromosome STS markers AZFa, AZFb and AZFc, ZFX/ZFY, and terminal sY160 regions. Y-chromosome STS markers were evaluated by DNA fragment analysis. 
 Results: The chart review indicated that a total of 319 men applied to our genetic diagnosis center between 2016 and 2020 due to infertility (mean age 32 ±7). Among the 319 infertile men, we determined 21 cases with Y chromosome microdeletions (6.89%), with the most common AZFc deletion (n=11, 52.3%) which is consistent with literature.
 Conclusion: Y-microdeletions are among the most common genetic causes of male infertility. In azoospermic and oligospermic patients, cytogenetic tests, Y-chromosome microdeletions and NGS panel screening tests can give effective information before the use of assisted reproductive techniques.

Y-chromosome haplogroups and Azoospermia Factor (AZF) analysis in Tunisian infertile male

The aim of the present study was to clarify the implication of Y chromosome genetic variations and haplogroups in Tunisian infertile men. A total of 27 Y-chromosomal binary markers partial microdeletions (gr/gr, b1/b3 and b2/b3) and copy number variation of DAZ and CDY genes in the AZFc region were analysed in 131 Tunisian infertile men with spermatogenic failure and severe reduced sperm concentrations and in 85 normospermic men as controls. Eleven different haplogroups in the overall population study (E3b2; J1J*, E1, E3b*, F, G, K, P/Q, R*, R1* and R1a1) were found. Interestingly, the J1J* haplogroup was significantly more frequent in azoo/oligospermic patients than in normospermic men (35.1% and 22.3%, respectively (p value = 0.04)). Results showed also that patients without DAZ/CDY1 copies loss and without partial microdeletions belonged to the R1 haplogroup. The relative high frequencies of two haplogroups, E3b2 (35.1%) and J (30%) was confirmed in Tunisia. We reported in the present study and for the first time, that J1J* haplogroup may confer a risk factor for infertility in the Tunisian population and we suggested that R1 haplogroup may ensure certain stability to Y-chromosome in Tunisian men.

Complete mitochondrial DNA sequence tries to settle hitherto putative history of Kayastha population of India.

Indian caste system is unique as it has an inimitable type of class system where the social ordering is done based on birth. Within the caste system, there is a distinct endogamous population known as the Kayastha, who have had inconclusive stratification records due to unidentified historical records. To gain a more inclusive view on the history and genetic affinities of Kayastha people, complete mitochondrial genomes from 15 individuals of a Kayastha population from North-western India have been sequenced. Interestingly, three novel sub-clades (U2b2a, M3d2, and M33a3b) have been identified that represent unique Kayastha motifs. The haplotype-based analysis suggests that the Kayastha population shares genetic affinities with the Indo-European and Sino-Tibetan populations found in the trans-Himalayan region. The FST based population comparison and the MDS plot indicates that Kayastha people have close maternal genetic affinity with the available genetic database of Brahmins, Kashmiris, and Tharus. The maternal genetic lineages among Kayastha population shows deep in situ origin that emerged much before settled life developed on this sub-continent. Both mtDNA and Y-chromosome markers, trace the genetic lineages of Kayastha population with Tharus, who regard themselves Kshatriya, corroborated by the oral history of the Kayasthas for their Kshatriya affiliation. It also validates genetic heritage of earliest settlers of India in both indigenous tribal and caste populations.

Структура генофонда томских татар по маркерам Y-хромосомы

Изучен генофонд томских татар, проживающих в трех населенных пунктах: д. Черная речка, д. Тахтамышево, д. Эушта. Выявлено, что томские татары являются самой гетерогенной группой на территории Сибири. Полученные данные о генофонде томских татар отражают сложный процесс его формирования на базе различных по происхождению предковых групп населения. По результатам анализа генетической дифференциации по частотам гаплогрупп при делении томских татар на три выборки в соответствии с населенными пунктами показана высокая степень их межгрупповых различий. Томские татары значительно отличаются от других групп сибирских татар по составу гаплогрупп. По разным гаплогруппам показана связь томских татар с телеутами, северными алтайцами, шорцами, хакасами, тувинцами и бурятами, что подтверждает их связь с южносибирскими тюркоязычными и монголоязычными народами. Также выявлена значительная доля генофонда, унаследованная от переселенцев из Поволжья и бухарцев из Средней Азии. The gene pool of Tomsk Tatars living in three settlements was studied: the village of Chernaya Rechka, the village of Takhtamyshevo, and the village of Eushta. It was revealed that the Tomsk Tatars are the most heterogeneous group in Siberia. The obtained results of the gene pool of the Tomsk Tatars reflect the complex process of their formation on the basis of ancestral population groups of different origins. According to the results of the analysis of genetic differentiation according to the frequencies of haplogroups, when dividing the Tomsk Tatars into three samples in accordance with the settlements, a high degree of their intergroup differences was shown. Tomsk Tatars differ significantly from other groups of Siberian Tatars in terms of the composition of haplogroups. According to different haplogroups, the connection of Tomsk Tatars with Teleuts, Northern Altaians, Shors, Khakass, Tuvans and Buryats is shown, which confirms their connection with the South Siberian Turkic-speaking and Mongolian-speaking peoples. A significant proportion of the gene pool inherited from people from the Volga region and Bukharians from Central Asia was also revealed.

Genetic ancestry in Afro-descendants from the Andes and Pacific Coast regions of Ecuador

This study aimed to investigate genetic diversity and ancestry in Afro-descendants from the Andes (AN) and the Pacific Coast (PC) regions of Ecuador, using autosomal and lineage markers. CR-mtDNA and PPY23-STRs showed high haplotype diversities. Statistically significant differences were found between AN and PC. Due to different patterns of sex-biased matting, differences between regions were more pronounced for the mtDNA than for the Y-chromosomal markers. A lower African ancestry was detected in AN than PC, for autosomal and both maternal and paternal lineage markers.

The effects of 21 cytological and nucleic acid binding dyes on autosomal and Y-chromosomal STR markers using new generation STR amplification kits

Before DNA fingerprinting analysis, many biochemical and histopathological testing are conducted on a forensic biological sample. The samples are exposed to cytological and nuclear binding dyes for their visualization under a microscope. The present study assessed the usability of these biological samples exposed to 21 routinely used histological and nuclear binding dyes for forensic DNA examination in a simulated experiment. Wright's stain, Hematoxylin, Coomassie Brilliant Blue, and Evan's Blue did not show any effect on the amplification of autosomal STR markers. Y-STRs showed higher robustness in presence of 10 dyes. Wright's stain showed the least effect on the generation of complete autosomal and Y-STR profiles. Mobility issues in capillary electrophoresis were observed in the presence of Eosin yellowish, Coomassie Brilliant Blue, and Hematoxylin for autosomal STRs and Wright's stain, Orcein, Malachite Green, Nigrosin, Bromophenol blue, and Fast green for Y STRs. Peak heights of the respective Y STR markers showed a mixed result in comparison to the positive control. In autosomal STR analysis, the peak heights showed a sharp decrease in comparison to the positive control.

Forensic Applications of Markers Present on the X Chromosome.

Microsatellite genetic markers are the gold standard for human genetic identification. Forensic analyses around the world are carried out through protocols using the analysis of STR markers in autosomal chromosomes and in the Y chromosome to solve crimes. However, these analyses do not allow for the resolution of all cases, such as rape situations with suspicion of incest, paternity without a maternal sample for comparison, and biological traces with DNA mixture where the profile sought is female, among other situations. In these complex cases, the study of X-chromosome STR markers significantly increases the probability of identification by complementing the data obtained for autosomal and Y-chromosome markers, due to the unique structure of the X chromosome and its exclusive method of inheritance. However, there are currently no validated Brazilian protocols for this purpose, nor are there any population data necessary for statistical analyses that must be included in the issuance of expert reports. Thus, the aim of this article is to provide a literary review of the applications of X-chromosomal markers in population genetics.

Development and validation of a new multiplex Y-STR panel designed to increase the power of discrimination.

In an attempt to increase the discrimination capacity (DC) and reduce the adventitious match probability, a 6-dye multiplex Y-chromosomal short tandem repeat (Y-STR) panel named Y34plex was constructed that combined 25 Y-chromosomal markers (DYS456, DYS627, DYS390, DYS570, DYS635, DYS385a/b, DYS448, DYS437, DYS533, DYS449, DYS481, DYS392, DYS391, DYS389I, DYS460, YGATAH4, DYS438, DYS389II, DYS19, DYS458, DYF387S1a/b, DYS439, DYS393, DYS576, and DYS518) in widely used commercial kits, with nine highly polymorphic Y-STR loci (DYS557, DYS527a/b, DYS593, DYS444, DYS596, DYS643, DYS447, DYS549, and DYS645). The Y34plex is a promising type system to distinguish both unrelated and related male individuals due to the incorporation of rapidly mutatedY-STR loci. A validation study of the Y34plex was performed and followed the guidelines of the Scientific Working Group on DNA analysis methods. Results show that full Y-STR profiles were obtained from male/female DNA mixtures with 125pg of male DNA in the presence of 50ng of female DNA. The ability to tolerate polymerase chain reaction inhibitors commonly contained in forensic casework samples demonstrated the applicability and robustness of the Y34plex. Compared with the Yfiler Plus kit, the novel panel showed an increased power of discrimination in Chinese Wuxi Han population (n=434). The overall haplotype diversity of the Y34plex was 0.999606, whereas DC value was 0.956221, which is suitable for use on forensic paternal investigation.

The development of an indel panel for microchimerism detection

ObjectivesThe aim of the study was to create a simple assay for microchimerism detection independent of sex and without HLA genotyping. MethodsThe method is based on detection of insertion or deletions utilizing a multiplex PCR followed by fragment analysis by capillary electrophoresis, and probe-based qPCR assays. A total of 192 samples, taken either before pregnancy, during 1st trimester, or either during 2nd trimester or at miscarriage, obtained from a cohort of 97 female patients with either primary or secondary recurrent pregnancy loss, were screened for fetal microchimerism by the indel panel as well as an existing assay based on detection of the Y-chromosome marker; DYS14. ResultsThe overall prevalence of DYS14 positive samples was 29% (55/192) whereas 32% (61/192) tested positive by the indel method. There was an overall agreement of 64% (122/192) between the results obtained by the two methods. A Fisher's Exact test showed no statistic significant difference in the prevalence of microchimerism detected by the two methods at any of the three times of sampling. The distribution of the number of positive wells detected by both methods were compared by a Mann-Whitney U test, which showed no statistically significant difference at any of the three times of sampling. ConclusionThe data indicates that microchimerism can be detected efficiently by the indel method. This makes it possible to detect both female and male cells without the need of HLA-genotyping. Furthermore, the indel method has potential to be implemented as a routine analysis. This will remove the sex bias in future explorations of the role microchimerism plays in health and disease.

Sequencing of autosomal, mitochondrial and Y-chromosomal forensic markers in the People of the British Isles cohort detects population structure dominated by patrilineages

Short tandem repeat (STR) polymorphisms are traditionally assessed by measuring allele lengths via capillary electrophoresis (CE). Massively parallel sequencing (MPS) reveals differences among alleles of the same length, thus improving discrimination, but also identifying groups of alleles likely related by descent. These may have relatively restricted geographical distributions and thus MPS could detect population structure more effectively than CE-based analysis. We addressed this question by applying an MPS multiplex, the Promega PowerSeq™ Auto/Mito/Y System prototype, to 362 individuals chosen to represent a wide geographical spread from the People of the British Isles (PoBI) cohort, which represents at least three generations of local rural ancestry. As well as 22 autosomal STRs (aSTRs; equivalent to PowerPlex Fusion loci) the system sequences 23 Y-STRs (the PowerPlexY 23 loci) and the control region (CR) of mitochondrial DNA (mtDNA), allowing population structure to be compared across biparentally and uniparentally inherited segments of the genome. For all loci, FST-based tests of population structure were done based on historical, linguistic, and geographical partitions, and for aSTRs the clustering algorithm STRUCTURE was also applied. STRs were considered using both length and sequence. Sequencing increased aSTR allele diversity by 87.5% compared to CE-based designations, reducing random match probability to 1.25E-30, compared to a CE-based 6.72E-27. Significant population structure was detectable in just one pairwise comparison (Central/South East England compared to the rest), and for sequence-based alleles only. The 362 samples carried 308 distinct mtDNA CR haplotypes corresponding to 13 broad haplogroups, representing a haplotype diversity of 0.9985 ( ± 0.0005), and a haplotype match probability of 0.0043. No significant population structure was observed. Y-STR haplotypes belonged to ten broad predicted Y-haplogroups. Allele diversity increased by 33% when considered at the sequence rather than length level, although haplotype diversity was unchanged at 0.999969 ( ± 0.000001); haplotype match probability was 2.79E-03. In contrast to the biparentally and maternally inherited loci, Y-STR haplotypes showed significant population structure at several levels, but most markedly in a comparison of regions subject to Anglo-Saxon influence in the east with the rest of the sample. This was evident for both length- and sequence-based allele designations, with no systematic difference between the two. We conclude that MPS analysis of aSTRs or Y-STRs does not generally reveal stronger population structure than length-based analysis, that UK maternal lineages are not significantly structured, and that Y-STR haplotypes reveal significant population structure that may reflect the Anglo-Saxon migrations to Britain in the 6th century.

Microhaplotype and Y-SNP/STR (MY): A novel MPS-based system for genotype pattern recognition in two-person DNA mixtures

BackgroundsY-chromosomal haplotypes based on Y-short tandem repeats (STRs) and Y-single nucleotide polymorphisms/insertion and deletion polymorphisms (SNPs/InDels) are used to characterize paternal lineages of unknown male trace donors. However, Y-chromosomal genetic markers are not currently sufficient for precise individual identification. Microhaplotype (MH), generally < 200 bp on autosomes and consisting of two or more SNPs, was recently introduced in forensic genetics with the development of massive parallel sequencing technology and may facilitate identification and DNA mixture deconvolution. Therefore, combining the two kinds of genetic markers may be beneficial in many forensic scenarios, especially crime scenes with male suspects, such as sexual assault cases. MethodsIn the present study, we developed a novel MPS-based panel, Microhaplotype and Y-SNP/STR (MY), by multiplex PCR and 150-bp paired-end sequencing, including 114 Y-SNPs (twelve dominant Y-DNA haplogroups), 45 Y-STRs (N-1 stutter < 0.09; estimated mutation rate < 5 × 10−3), and 22 MHs (allele coverage ratio > 0.91; pairwise distance > 10 Mb). Additionally, MY system-based genotype pattern recognition (GPR), a regression-based method to identify the genotype pattern for each MH locus, is proposed for two-person DNA mixture deconvolution. We integrated 26 two-person genotype combinations into nine genotype patterns and validated the application range of GPR based on DNA profiles of ten sets of simulated male-male DNA mixtures (1:10–1:2). ResultsThe effective number of alleles (Ae) ranged from 3.62 to 14.72, with an average of 7.17, in 100 Chinese Guangdong Han individuals. The cumulative discrimination power was 1–5.00 × 10−31, and the cumulative power of exclusion was 1–5.00 × 10−8 and 1–4.85 × 10−12 for duo and trio paternity testing, respectively. Furthermore, the actual mixing ratio-depth of coverage (DoC) ratio (RDoC) regression relationships were established for different genetic markers and genotype patterns. In five overlapping areas, genotype differentiation of the major and minor contributors required likelihood ratio methods. In nonoverlapping areas, the genotype pattern could be recognized by comparing the observed RDoC and RDoC ranges. ConclusionThe GPR can be used to deconvolute two-person DNA mixtures (application range: 1:10–1:2) for individual identification.