Development and validation of a custom panel including 256 Y-SNPs for Chinese Y-chromosomal haplogroups dissection

Y-chromosomal haplogroups determined by Y-chromosomal single nucleotide polymorphisms (Y-SNPs) allow paternal lineage identification and paternal biogeographic ancestry inference, which has attracted a lot of interest in the forensic community. Recently, a comprehensive Y-SNP tool with dominant markers targeting haplogroups in R, E and I branches has been reported, which allows the inference of 640 Y haplogroups. It had a very good performance and could provide a high level of Y haplogroup resolution in most populations. However, the predominant haplogroups in the Chinese populations are O, C and N, suggesting that more Y-SNPs under these clades are needed to achieve the population-specific high resolution. Herein, aiming at the Chinese population, we presented a largely improved custom Y-SNP MPS panel that contains 256 carefully ascertained Y-SNPs based on our previous studies, and evaluated this panel via a series of tests, including the tests for concordance, repeatability, sensitivity, specificity, and stability, as well as the mixture, degraded and case-type sample analysis. The preliminary developmental validation demonstrated that this panel was highly reliable, sensitive, specific, and robust. In the sensitivity test, even when the DNA input was reduced to as low as 0.5 ng, the sample could still be assigned to the correct Y haplogroup. For mixture analysis, even the 1:99 (Male: Female) mixtures had no effects on the assignation of the Y haplogroup of the male contributor. In summary, this assay has provided a high-resolution Y-chromosomal haplogrouping workflow to determine a male’s paternal lineage and/or paternal biogeographic ancestry and could be widely used for Chinese Y-chromosomal haplogroups dissection.     

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.     

Developmental validation of a 381 Y-chromosome SNP panel for haplogroup analysis in the Chinese populations

Y-chromosome single nucleotide polymorphism (Y-SNP) shows great variation in geographical distribution and population heterogeneity and can be used to map population genetics around the world. Massive parallel sequencing (MPS) methodology enables high-resolution Y-SNP haplogrouping for a certain male and is widely used in forensic genetics and evolutionary studies. In this present study, we used MPS to develop a customized 381 Y-SNP panel (SifaMPS 381 Y-SNP panel) to investigate the basic structure and subbranches of the haplogroup tree of the Chinese populations. The SifaMPS 381 Y-SNP panel covers all the Y-SNPs from our previously designed 183 Y-SNP panel and additional SNPs under the predominant haplogroups in the Chinese populations based on certain criteria. We also evaluated the sequencing matrix, concordance, sensitivity, repeatability of this panel and the ability to analyze mixed and case-type samples based on the Illumina MiSeq System. The results demonstrated that the novel MPS Y-SNP panel possessed good sequencing performance and generated accurate Y-SNP genotyping results. Although the recommended DNA input was greater than 1.25 ng, we observed that a lower DNA amount could still be used to analyze haplogroups correctly. In addition, this panel could handle mixed samples and common case-type samples and had higher resolution among Chinese Han males than previously reported. In conclusion, the SifaMPS 381 Y-SNP panel showed an overall good performance and offers a better choice for Y-SNP haplogrouping of the Chinese population, thereby facilitating paternal lineage classification, familial searching and other forensic applications.     

Testing the Ion AmpliSeq™ HID Y-SNP Research Panel v1 for performance and resolution in admixed South Americans of haplogroup Q

Y haplogroups, defined by Y-SNPs, allow the reconstruction of the human Y chromosome genealogy, which is important for population, evolutionary and forensic genetics. In this study, Y-SNPs were typed and haplogroups inferred with the MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1, as a high-throughput approach. Firstly, the performance of the panel was evaluated with different DNA input amounts, reagent volumes and cycle numbers. DNA-inputs from 0.5 to 1 ng generated the most balanced read depth. Combined with full reagent and 19 cycles, this offered the highest number of amplicons with a sequencing read depth of at least 20 reads. Secondly, the sub-haplogroups of 182 admixed South Americans and Greenlanders belonging to haplogroup Q were inferred and tested for potential improvement in resolution. Most samples were assigned to lineage Q-M3 with some samples assigned to lineages upstream (Q-M346, L56, L57; Q-L331, L53; Q-L54; Q-CTS11969, CTS11970) or parallel (Q-L330, L334; Q-Z780/M971) to Q-M3. Only one sample was assigned to a downstream lineage (Q-Z35615, Z35616). Most individuals of haplogroup Q with NAM ancestry could neither be distinguished from each other, nor from half of the Greenlandic samples. Typing additional, known SNPs within lineage Q-M3, Z19483 and SA05, increased the resolution of predicted haplogroups. The search for novel variants in the sequenced regions allowed the detection of 42 variants and the subdivision of lineage Q-M3 into new subclades. The variants found in six of these subclades were exclusive to certain South American countries. In light of the limited differentiation of haplogroup Q samples, the additional information on known or novel SNPs disclosed in this study when using MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1 should be included in the Yleaf software, to increase the differentiation of lineage Q-M3.     

Developmental validation of a custom panel including 165 Y-SNPs for Chinese Y-chromosomal haplogroups dissection using the ion S5 XL system

Massively parallel sequencing (MPS) technology has the capability to genotype hundreds to thousands of SNPs from multiple samples, with high coverage in a single experimental run. MPS, which enables high-resolution Y haplogrouping from limited DNA resources, has attracted much interest in Y-chromosomal applications, particularly human forensics and anthropological studies. Previously, we selected and designed a 72 Y-SNPs MPS panel aiming at the Chinese population for providing the basic structure of the haplogroup tree, and then additional Y-SNPs were screened out for further subdivision into different populations by using pyrosequencing technology. In the present study, we designed a customized MPS panel that includes 165 Y-SNPs based on our previous studies and evaluated the sequencing performance, sensitivity and the ability of the MPS-SNP panel to analyze case-type samples using the Ion S5 XL System. In the concordant study, only four Y-SNPs presented missing genotypes due to low coverage reads, and five SNPs were prone to being designated heterozygotes, whereas the designation of the other SNPs were fully consistent with the results obtained from other Y-SNP typing tools. The analyst-interpreted Y-SNP profiles demonstrated that the targeted Y-SNPs, except for the nine poorly performing Y-SNPs, performed well and could acquire achievable high-resolution. Moreover, this MPS panel could be adapted to case-type samples, and single source complete profiles could be stably obtained using 1 ng of input DNA. In summary, this MPS Y-SNP panel offers a straightforward sample-to-haplogroup workflow that would be beneficial for paternal lineage classification and forensic pedigree searches, and the present results provide support for the application of MPS technology in SNP analysis.     

An efficient multiplex genotyping approach for detecting the major worldwide human Y-chromosome haplogroups

The Y chromosome is paternally inherited and therefore serves as an evolutionary marker of patrilineal descent. Worldwide DNA variation within the non-recombining portion of the Y chromosome can be represented as a monophyletic phylogenetic tree in which the branches (haplogroups) are defined by at least one SNP. Previous human population genetics research has produced a wealth of knowledge about the worldwide distribution of Y-SNP haplogroups. Here, we apply previous and very recent knowledge on the Y-SNP phylogeny and Y-haplogroup distribution by introducing two multiplex genotyping assays that allow for the hierarchical detection of 28 Y-SNPs defining the major worldwide Y haplogroups. PCR amplicons were kept small to make the method sensitive and thereby applicable to DNA of limited amount and/or quality such as in forensic settings. These Y-SNP assays thus form a valuable tool for researchers in the fields of forensic genetics and genetic anthropology to infer a man's patrilineal bio-geographic ancestry from DNA.     

Defining Y-SNP variation among the Flemish population (Western Europe) by full genome sequencing

Y-chromosomal single nucleotide polymorphisms (Y-SNPs) represent a powerful tool in forensic research and casework, especially for inferring paternal ancestry of unknown perpetrators and unidentified bodies. However, the wealth of recently discovered Y-SNPs, the ‘jungle’ of different evolutionary lineage trees and nomenclatures, and the lack of population-wide data of many phylogenetically mapped Y-SNPs, limits the use of Y-SNPs in routine forensic approaches. Recently, a concise reference phylogeny of the human Y chromosome, the ‘Minimal Reference Y-tree’, was introduced aiming to provide a stable phylogeny with optimal global discrimination capacity by including the most resolving Y-SNPs. Here, we obtained a representative sample of 270 whole-genome sequences (WGS) to grasp the Y-SNP variation within the autochthonous Flemish population (Belgium, Western Europe) according to this reference Y-tree. The high quality of the Y-SNP calling was guaranteed for the WGS sample as well as its representativeness for the Flemish population based on the comparison of the main haplogroup frequencies with those from earlier studies on Flanders and the Netherlands. The 270 Flemish Y chromosomes were assigned to 98 different sub-haplogroups of the Minimal Reference Y-tree, showing its high potential of discrimination and confirming the spectrum of evolutionary lineages within Western Europe in general and within Flanders in particular. The full database with all Y-SNP calls of the Flemish sample is public available for future updates including forensic and population genetic studies. New initiatives to categorise Y-SNP variation in other populations according to the reference phylogeny of the Y chromosome are highly encouraged for forensic applications. Recommendations to realise such future population sample sets are discussed based on this study.     

Development and inter-laboratory evaluation of the VISAGE Enhanced Tool for Appearance and Ancestry inference from DNA

Responding to the growing scientific and practical interest in forensic DNA phenotyping, the VISible Attributes through GEnomics (VISAGE) Consortium was founded in 2017 with the main goal of developing and validating new and reliable molecular and statistical tools to predict appearance, ancestry and age from DNA. Here, we describe the development and inter-laboratory evaluation and validation of the VISAGE Enhanced Tool for Appearance and Ancestry inference from DNA. The VISAGE Enhanced Tool for Appearance and Ancestry is the first forensic-driven genetic laboratory tool that comprises well-established markers for eye, hair and skin color with more recently discovered DNA markers for eyebrow color, freckling, hair shape and male pattern baldness and bio-geographic ancestry informative DNA markers. The bio-geographic ancestry markers include autosomal SNPs (bi- and tri-allelic SNPs), X-SNPs, Y-SNPs and autosomal Microhaplotypes. In total, primers targeting 524 SNPs (representing a 97.6% assay conversion rate) were successfully designed using AmpliSeq into a single primer pool (i.e., one multiplex assay) and sequenced with the Ion S5. In a collaborative framework, five VISAGE laboratories tested the VISAGE Enhanced Tool for Appearance and Ancestry on reproducibility, sensitivity, genotyping concordance, mixtures, species specificity and performance in relevant forensic conditions, including inhibitor-spiked, mock casework and artificially degraded samples. Based on our results, the VISAGE Enhanced Tool for Appearance and Ancestry is a robust, reproducible, and – for the large SNP number - fairly sensitive MPS assay with high concordance rates. With the VISAGE Enhanced Tool for Appearance and Ancestry introduced here, the VISAGE Consortium delivers the first single DNA-test for combined appearance prediction based on seven traits together with bio-geographic ancestry inference based on major continental regions for separated bi-parental and paternal ancestry, which represents the most comprehensive validated laboratory tool currently available for Forensic DNA Phenotyping.     

Improving the regional Y-STR haplotype resolution utilizing haplogroup-determining Y-SNPs and the application of machine learning in Y-SNP haplogroup prediction in a forensic Y-STR database: A pilot study on male Chinese Yunnan Zhaoyang Han population

Improving the resolution of the current widely used Y-chromosomal short tandem repeat (Y-STR) dataset is of great importance for forensic investigators, and the current approach is limited, except for the addition of more Y-STR loci. In this research, a regional Y-DNA database was investigated to improve the Y-STR haplotype resolution utilizing a Y-SNP Pedigree Tagging System that includes 24 Y-chromosomal single nucleotide polymorphism (Y-SNP) loci. This pilot study was conducted in the Chinese Yunnan Zhaoyang Han population, and 3473 unrelated male individuals were enrolled. Based on data on the male haplogroups under different panels, the matched or near-matching (NM) Y-STR haplotype pairs from different haplogroups indicated the critical roles of haplogroups in improving the regional Y-STR haplotype resolution. A classic median-joining network analysis was performed using Y-STR or Y-STR/Y-SNP data to reconstruct population substructures, which revealed the ability of Y-SNPs to correct misclassifications from Y-STRs. Additionally, population substructures were reconstructed using multiple unsupervised or supervised dimensionality reduction methods, which indicated the potential of Y-STR haplotypes in predicting Y-SNP haplogroups. Haplogroup prediction models were built based on nine publicly accessible machine-learning (ML) approaches. The results showed that the best prediction accuracy score could reach 99.71% for major haplogroups and 98.54% for detailed haplogroups. Potential influences on prediction accuracy were assessed by adjusting the Y-STR locus numbers, selecting Y-STR loci with various mutabilities, and performing data processing. ML-based predictors generally presented a better prediction accuracy than two available predictors (Nevgen and EA-YPredictor). Three tree models were developed based on the Yfiler Plus panel with unprocessed input data, which showed their strong generalization ability in classifying various Chinese Han subgroups (validation dataset). In conclusion, this study revealed the significance and application prospects of Y-SNP haplogroups in improving regional Y-STR databases. Y-SNP haplogroups can be used to discriminate NM Y-STR haplotype pairs, and it is important for forensic Y-STR databases to develop haplogroup prediction tools to improve the accuracy of biogeographic ancestry inferences.     

Y chromosome haplogroup diversity in a Mestizo population of Nicaragua

Y chromosome single nucleotide polymorphisms (Y-SNPs) are indispensable markers for haplogroup determination. Since Y chromosome haplogroups show a high specific geographical distribution, they play a major role in population genetics but can also benefit forensic investigations. Although haplogroup prediction methods based on Y chromosome short tandem repeats (Y-STRs) exist and are frequently used, precaution is required in this regard. In this study we determine the Y chromosome haplogroups of a Nicaraguan population using several Y-SNP multiplex reactions. Y chromosome haplogroups have been predicted before, but our results show that a confirmation with Y-SNP typings is necessary. These results have revealed a 4.8% of error in haplogroup prediction based on Y-STR haplotypes using Athey's Haplogroup Predictor. The Nicaraguan Mestizo population displays a majority of Eurasian lineages, mainly represented by haplogroup R-M207 (46.7%). Other Eurasian lineages have been observed, especially J-P209 (13.3%), followed by I-M170 (3.6%) and G-M201 (1.8%). Haplogroup E-P170 was also observed in 15.2% of the sample, particularly subhaplogroup E1b1b1-M35. Finally, the Native American haplogroup Q-M242 was found in 15.2% of the sample, with Q1a3a-M3 being the most frequent.     

A one step multiplex PCR assay for rapid screening of East Asian mtDNA haplogroups on forensic samples

The mitochondrial DNA (mtDNA) haplogroup typing has become an essential tool to study human evolutionary history and to infer the matrilineal bio-geographic ancestry. In forensic field, the screening of mtDNA haplogroups by genotyping of mtDNA single nucleotide polymorphisms (SNPs) can help guarantee the quality of mtDNA sequence data as well as can reduce the need to sequence samples that do not match. Here, a multiplex mutagenically separated (MS) polymerase chain reaction (PCR) system was developed for simultaneous rapid detection of 14 coding region SNPs and one deletion motif representing common mtDNA haplogroups of East Asia. The multiplex MS PCR system we developed has the advantage of being a one step procedure that requires only a single PCR amplification with allele-specific primers and allowing straightforward designation of haplogroups along the branches of the phylogenetic tree. Therefore, it would be a simple, rapid, and reliable detection method useful for large-scale screening of mtDNA variations to determine East Asian mtDNA haplogroups.     

Haplogrouping mitochondrial DNA sequences in Legal Medicine/Forensic Genetics

Haplogrouping refers to the classification of (partial) mitochondrial DNA (mtDNA) sequences into haplogroups using the current knowledge of the worldwide mtDNA phylogeny. Haplogroup assignment of mtDNA control-region sequences assists in the focused comparison with closely related complete mtDNA sequences and thus serves two main goals in forensic genetics: first is the a posteriori quality analysis of sequencing results and second is the prediction of relevant coding-region sites for confirmation or further refinement of haplogroup status. The latter may be important in forensic casework where discrimination power needs to be as high as possible. However, most articles published in forensic genetics perform haplogrouping only in a rudimentary or incorrect way. The present study features PhyloTree as the key tool for assigning control-region sequences to haplogroups and elaborates on additional Web-based searches for finding near-matches with complete mtDNA genomes in the databases. In contrast, none of the automated haplogrouping tools available can yet compete with manual haplogrouping using PhyloTree plus additional Web-based searches, especially when confronted with artificial recombinants still present in forensic mtDNA datasets. We review and classify the various attempts at haplogrouping by using a multiplex approach or relying on automated haplogrouping. Furthermore, we re-examine a few articles in forensic journals providing mtDNA population data where appropriate haplogrouping following PhyloTree immediately highlights several kinds of sequence errors.     

Evaluation of Y chromosomal SNP haplogrouping in the HID-Ion AmpliSeq™ Identity Panel

The Y chromosomal haplogroup determined from single nucleotide polymorphism (SNP) combinations is a valuable genetic marker to study ancestral male lineage and ethical distribution. Next-generation sequencing has been developed for widely diverse genetics fields. For this study, we demonstrate 34 Y-SNP typing employing the Ion PGM™ system to perform haplogrouping. DNA libraries were constructed using the HID-Ion AmpliSeq™ Identity Panel. Emulsion PCR was performed, then DNA sequences were analyzed on the Ion 314 and 316 Chip Kit v2. Some difficulties became apparent during the analytic processes. No-call was reported at rs2032599 and M479 in six samples, in which the least coverage was observed at M479. A minor misreading occurred at rs2032631 and M479. A real time PCR experiment using other pairs of oligonucleotide primers showed that these events might result from the flanking sequence. Finally, Y haplogroup was determined completely for 81 unrelated males including Japanese (n = 59) and Malay (n = 22) subjects. The allelic divergence differed between the two populations. In comparison with the conventional Sanger method, next-generation sequencing provides a comprehensive SNP analysis with convenient procedures, but further system improvement is necessary.     

Understanding the Y chromosome variation in Korea—relevance of combined haplogroup and haplotype analyses

We performed a molecular characterization of Korean Y-chromosomal haplogroups using a combination of Y-chromosomal single nucleotide polymorphisms (Y-SNPs) and Y-chromosomal short tandem repeats (Y-STRs). In a test using DNA samples from 706 Korean males, a total of 19 different haplogroups were identified by 26 Y-SNPs including the newly redefined markers (PK4, KL2, and P164) in haplogroup O. When genotyping the SNPs, phylogenetic nonequivalence was found between SNPs M117 and M133, which define haplogroup O3a3c1 (O3a2c1a according to the updated tree of haplogroup O by Yan et al. (European Journal of Human Genetics 19:1013-1015, 2011)), suggesting that the position of the M133 marker should be corrected. We have shown that the haplotypes consisted of DYS392, DYS393, DYS437, DYS438, DYS448, and DYS388 loci, which exhibit a relatively lower mutation rate, can preserve phylogenetic information and hence can be used to roughly distinguish Y-chromosome haplogroups, whereas more rapidly mutating Y-STRs such as DYS449 and DYS458 are useful for differentiating male lineages. However, at the relatively rapidly mutating DYS447, DYS449, DYS458, and DYS464 loci, unusually short alleles and intermediate alleles with common sequence structures are informative for elucidating the substructure within the context of a particular haplogroup. In addition, some deletion mutations in the DYS385 flanking region and the null allele at DYS448 were associated with a single haplogroup background. These high-resolution haplogroup and haplotype data will improve our understanding of regional Y-chromosome variation or recent migration routes and will also help to infer haplogroup background or common ancestry.     

Hierarchical Y-SNP assay to study the hidden diversity and phylogenetic relationship of native populations in South America

Studying the Y chromosomes of indigenous tribes of Ecuador revealed a lack of strategic SNP assays to examine the substructure of South American native populations. In most studies dealing with South American samples so far only the most common Y-SNP M3 of haplogroup Q was analyzed, because this is known to define a founder group in South America. Studies of SNPs ancestral to Q-M3 (Q1a3a) to confirm the results or the typing of Q subclades have often been neglected. For this reason we developed a SNaPshot assay, which allows first for a hierarchical testing of all main haplogroups occurring in South American populations and second for a detailed analysis of haplogroups Q and C thought having ancient Asian descent. We selected 16 SNPs from the YCC haplogroup tree and established two multiplexes. The first multiplex ("SA Major") includes 12 Y-SNPs defining the most frequent haplogroups occurring in South America (M42, M207, M242, M168, M3, M145, M174, M213, RPS4Y711, M45, P170, and M9). The second multiplex ("SA SpecQ") contains Y-SNPs of haplogroup Q, especially of the subclade Q-M3 (M19, M194, P292, M3, and M199). Within our Ecuadorian sample, haplogroup Q-M3 (xM19, M194, P292, and M199) was predominant, but we also found haplogroup E and R, which can be attributed to recent admixture. Moreover, we found four out of 65 samples, which were tested to be haplogroup C3* (C-M217) the modal haplogroup in Mongolians and widespread in indigenous populations of the Russian Far East as well as in Eastern Asia. This haplogroup is not known to be the result of recent admixture and has been found only one time before in South America. Since haplogroup C occurs in Asia and in North America (C3b or C-P39), we assume that these C-lineages are ancient as well. Therefore, we established a third multiplex ("SA SpecC"), which allows the further subtyping of haplogroup C, mainly of subclade C3 defined by the Y-SNP M217 (M407, M48, P53.1, M217, P62, RPS4Y711, M93, M86, and P39). Altogether, these three multiplexes cover the most frequent haplogroups in South America and allow for a maximal resolution of the Y-chromosomal SNP diversity in Amerindian population samples.     

Separate analysis of DYS385a and b versus conventional DYS385 typing: is there forensic relevance?

In order to determine to what extent the separate analysis of both copies of DYS385 improves Y-chromosomal short tandem repeat (Y-STR) haplotyping, we followed a recently published protocol for the separate amplification of DYS385a and DYS385b with modifications and compared the results with those obtained by conventional analysis in a population sample comprising 133 unrelated Caucasian males from Austria. Additionally, we typed all markers of the minimal haplotype (minHT) and a set of Y-chromosomal single nucleotide polymorphisms (Y-SNPs) in order to interpret the STR data depending on the Y-SNP haplogroup structure. The separate amplification of DYS385a and b improved the power of discrimination of this marker when compared to the results obtained with the conventional non-locus-discriminating amplification strategy. However, the degree of this improvement varied greatly between different haplogroups and was found to be highest in clade K. In the forensically relevant context of the minHT, the separate analysis of the DYS385 alleles had no effect on the differentiation of paternal lineages in our study. Furthermore, the amplicon lengths of 700–780 base pairs obtained in the course of the locus-discriminating approach restrict the applicability of this amplification strategy to high quality DNA samples.     

A custom hybridisation enrichment forensic intelligence panel to infer biogeographic ancestry,hair and eye colour,and Y chromosome lineage

Massively parallel sequencing can provide genetic data for hundreds to thousands of loci in a single assay for various types of forensic testing. However, available commercial kits require an initial PCR amplification of short-to-medium sized targets which limits their application for highly degraded DNA. Development and optimisation of large PCR multiplexes also prevents creation of custom panels that target different suites of markers for identity, biogeographic ancestry, phenotype, and lineage markers (Y-chromosome and mtDNA). Hybridisation enrichment, an alternative approach for target enrichment prior to sequencing, uses biotinylated probes to bind to target DNA and has proven successful on degraded and ancient DNA. We developed a customisable hybridisation capture method, that uses individually mixed baits to allow tailored and targeted enrichment to specific forensic questions of interest. To allow collection of forensic intelligence data, we assembled and tested a custom panel of hybridisation baits to infer biogeographic ancestry, hair and eye colour, and paternal lineage (and sex) on modern male and female samples with a range of self-declared ancestries and hair/eye colour combinations. The panel correctly estimated biogeographic ancestry in 9/12 samples (75%) but detected European admixture in three individuals from regions with admixed demographic history. Hair and eye colour were predicted correctly in 83% and 92% of samples respectively, where intermediate eye colour and blond hair were problematic to predict. Analysis of Y-chromosome SNPs correctly assigned sex and paternal haplogroups, the latter complementing and supporting biogeographic ancestry predictions. Overall, we demonstrate the utility of this hybridisation enrichment approach to forensic intelligence testing using a combined suite of biogeographic ancestry, phenotype, and Y-chromosome SNPs for comprehensive biological profiling.     

Resolving mitochondrial haplogroups B2 and B4 with next-generation mitogenome sequencing to distinguish Native American from Asian haplotypes

Mitochondrial haplogroup information can be useful in forensic contexts that rely primarily on mitochondrial DNA (mtDNA) testing, which often involve limited or degraded DNA. Due to the phylogeographic patterning of mtDNA in human populations, mitochondrial haplogroups are indicative of maternal ancestry (as mtDNA is a maternally inherited marker). In certain circumstances, maternal ancestry inferred from mitochondrial haplogrouping could be beneficial to forensic investigations. For example, ancestry information could assist in the identification of unknown service members from past conflicts, such as the World War II Battle of Tarawa involving American and Japanese forces. In this context, it could be useful to distinguish Native American mtDNA from Asian mtDNA to bolster the anthropological and circumstantial evidence leading to an identification or foreign national determination. Although most of the founding Native American haplogroups contain diagnostic variants in the mitochondrial control region (CR), haplogroup B2 does not, and this makes it more difficult to distinguish B2 from the parental B4 and closely related B4b haplogroups found in Asia. In this paper, the amount of mtDNA information required to distinguish Native American haplotypes from Asian haplotypes within haplogroup B was examined. Fifty-six samples belonging to subtypes of B2 and B4 were sequenced for the entire mitogenome. Haplogroups were estimated from three ranges of mitochondrial DNA (HV1 and 2, CR, and full mitogenome). Half of the samples could not be precisely haplogrouped without full mitogenome data, although enough variants were often provided to make an accurate B2 versus B4 distinction. Native American B2 haplotypes were distinguishable using CR data alone in 82% of samples, though the remaining samples required full mitogenome data for haplogroup B2 designation. The use of full mitogenome data consistently enables accurate haplogroup determination, and opens the possibility for gaining information on maternal ancestry.     

A 16-plex Y-SNP typing system based on allele-specific PCR for the genotyping of Chinese Y-chromosomal haplogroups

Y-chromosomal SNP (Y-SNP), with its stable inheritance and low mutation, can provide Supplementary information in forensic investigation. While commonly used Y-chromosomal STR haplotypes show their limitations, typing of Y-SNP would become a powerful complement. In this study, a 16-plex Y-SNP typing system based on allele-specific PCR (AS-PCR) was developed to discriminate four dominant Y-chromosomal haplogroups (C-M130, D-CTS3946, N-M231, and O-M175) and 12 predominant sub-haplogroups of O-M175 (O1a-M119, O1a1a1a-CTS3265, O1b-M268, O1b1a2-Page59, O2-M122, O2a1-L127.1, O2a1b-F240, O2a1b1a1-CTS5820, O2a2-P201, O2a2b1a1-M177, O2a2b1a1a1a-Y17728, O2a2b1a2-F114). A series of experimental validation studies including sensitivity, species specificity, male-female mixture and inhibition were performed. The discrimination of the typing system was preliminarily proved with a haplogroup diversity of 0.9239. Altogether, the Y-SNP typing system based on AS-PCR should be capable of distinguishing China’s dominant Y-chromosomal haplogroups in a rapid and reliable manner, thus can be employed as a useful complement in forensic casework.     

Multiple recurrent mutations at four human Y-chromosomal single nucleotide polymorphism sites in a 37 bp sequence tract on the ARSDP1 pseudogene

The male-specific region of the human Y chromosome (MSY) is passed down clonally from father to son and mutation is the single driving force for Y-chromosomal diversification. The geographical distribution of MSY variation is non-random. Therefore, Y-chromosomal single nucleotide polymorphisms (Y-SNPs) are of forensic interest, as they can be utilized, e.g. for deducing the bio-geographical origin of biological evidence. This extra information can complement short tandem repeat data in criminal investigations. For forensic applications, however, any targeted marker has to be unequivocally interpretable.Here, we report findings for 17 samples from a population study comprising specimens from ∼3700 men living in Tyrol (Austria), indicating apparent homoplasic mutations at four Y-SNP loci on haplogroup R-M412/L51/S167, R-U152/S28, and L-M20 Y chromosomes. The affected Y-SNPs P41, P37, L202, and L203 mapped to a 37 bp region on Yq11.21. Observing in multiple phylogenetic contexts up to four homoplasic mutations within such a short sequence tract is unlikely to result from a series of independent parallel mutations. Hence, we rather propose X-to-Y gene conversion as a more likely scenario.Practical implications arising from markers exhibiting paralogues on the Y chromosome or sites with a high propensity to recurrent mutation for database searches are addressed.