Y-SNP miniplexes for East Asian Y-chromosomal haplogroup determination in degraded DNA

doi:10.1016/j.fsigen.2012.06.014

Forensic Science International: Genetics

Volume 7, Issue 1, January 2013, Pages 75-81

https://doi.org/10.1016/j.fsigen.2012.06.014 Get rights and content

Abstract

Four multiplex PCR systems followed by single base extension reactions were developed to score 22 single nucleotide polymorphisms (SNPs) and identify the most frequent East Asian Y chromosome haplogroups. Select Y chromosome SNPs allowed hierarchical testing for almost all of the major East Asian haplogroups along the revised Y chromosome tree. The first multiplex consists of six SNPs defining world-wide major haplogroups (M145, RPS4Y₇₁₁, M89, M9, M214, and M175). The second multiplex includes six SNPs of subhaplogroup O (M119, P31, M95, SRY₄₆₅, 47z, and M122). The third multiplex contains six SNPs that subdivide the subhaplogroup O3 (M324, P201, M159, M7, M134, and M133). The fourth multiplex comprises four SNPs of subhaplogroup C (M217, M48, M407, and P53.1). The sizes of the PCR amplicons ranged from 70 to 100 bp to facilitate their application to degraded forensic and ancient samples. Validation experiments demonstrated that the multiplexes were optimized for analysis of low template DNA and highly degraded DNA. In a test using DNA samples from 300 Korean males, 16 different Y chromosome haplogroups were identified; haplogroup O2b* was the most frequently observed (29.3%), followed by haplogroups C3 (xC3c, C3d, C3e) (16.0%) and O3a3c1 (11.0%). These multiplex sets will be useful tools for Y-chromosomal haplogroup determination in anthropological and forensic studies of East Asian populations.

Introduction

Genetic variations in the non-recombining portion of the Y chromosome (NRY) are analyzed in diverse disciplines including anthropological, forensic and medical genetics [1], [2], [3], [4], [5]. Because of a lack of recombination and low mutation rates, Y chromosome single nucleotide polymorphisms (Y-SNPs) are the most useful genetic markers for reconstructing male lineages. Therefore, Y chromosome haplogroups, which are defined by combinations of allelic states at hierarchically arranged Y-SNPs and small indels, have been extensively studied to infer the origins, evolution, and histories of migrations of modern human populations [6], [7].

A number of changes have been made to the Y-chromosomal haplogroup tree, and a total of 311 distinct haplogroups have been defined with increased resolution [8]. The haplogroup O was considerably rearranged during this revision; the L1 retroposon insertion (LINE 1) polymorphism, which had conflicted with the N7 polymorphism, was excluded from the list of markers used to define subhaplogroup O3. Persistent commercial or in-house development efforts for Y-SNP typing protocols have been made, but most developed methods involve typing of European Y haplogroups or world-wide major haplogroups with low resolution [9], [10], [11], [12] and are not suitable to subdivide subhaplogroup O3, a major haplogroup in East Asians [13], [14], into internal derivatives following the revised Y haplogroup tree. In addition, as the demand for inferring geographic origin is increasing in forensic DNA analysis as well as in ancient DNA analysis (e.g., identification of Korean War and Vietnam War victims, and genetic characterization of ancient remains), the development of sensitive and efficient methods for the Y-chromosomal haplogroup determination in degraded DNA is required.

Therefore, in the current study, a multiplex single base extension (SBE) method was developed to score Y-SNPs of East Asian haplogroups following a small size amplicon strategy that is suitable for application to degraded DNA. Y-SNPs were selected from SNPs that are hierarchically located along the revised topology of Y chromosome haplogroups, focusing on East Asian-specific haplogroups. To assess the utility of our method for the analysis of highly degraded samples, the sensitivity and efficiency of the multiplex set were validated in samples of serially diluted DNA, artificially degraded DNA, and DNA extracted from 55-year-old skeletal remains. Finally, a Korean sample was analyzed using the newly developed multiplex systems, and the distribution of Y haplogroups was studied, since the geographic origins and history of migration of a population can be inferred from the distributions and ages of its haplogroups [13], [14], [15].

Section snippets

DNA samples

Our study protocol was approved by the Institutional Review Board of Severance Hospital, Yonsei University in Seoul, Korea. DNA samples from 300 unrelated Korean males were obtained from the National Biobank of Korea. DNA concentrations were measured using a NanoDrop^® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the final sample concentrations were adjusted to 1.0 ng/μl. For the sensitivity test, 9948 male DNA (Promega, Madison, WI, USA) was serially diluted to

Y-SNP selection and primer design

The selected 22 Y-SNP markers were amplified with four multiplex PCR systems to explore East Asian Y chromosome haplogroups. Multiplex I was composed of the six Y-SNPs, M145, RPS4Y₇₁₁, M89, M9, M214, and M175, which distinguish all world-wide major clades except for the African lineages A and B. Among the six major haplogroups in Multiplex I, clade O is the most common haplogroup in East Asians, and accordingly, a more detailed analysis of the haplogroup O lineage is necessary to differentiate

Conclusion

Four multiplex systems for scoring 22 Y-SNPs were developed to identify the most frequent East Asian Y-chromosomal haplogroups, and subsequent validation tests showed that the multiplexes are sensitive and efficient for analyzing low template and highly degraded DNA. The newly developed multiplexes allowed not only subdivision of haplogroup C, but also subdivision of haplogroup O and its derived subhaplogroup O3 according to the revised Y chromosome tree, thereby demonstrating their suitability

Acknowledgements

The biospecimens for this study were provided by National Biobank of Korea, Korea Centers for Disease Control and Prevention, supported by the Korean Ministry of Health and Welfare. This study was supported by a faculty research grant from Yonsei University College of Medicine in Seoul, Korea for 2008 (6-2008-0266).

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All but M172 marker that was analyzed by direct sequencing were analyzed using in-house multiplex PCR and SBE reaction. The detailed methods for determination of Y chromosomal haplogroup are described in the Supplementary Material. In addition, genealogical relationships among haplotypes of the Pathan population and Jewish populations [21,22] within haplogroups G and Q were reconstructed using the program Network 4.6.1.1 [23].
We analyzed haplotypes for 22 Y chromosomal STRs (Y-STRs), including 17 Yfiler loci (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DY438, DYS439, DYS448, DYS456, DYS458, DYS635 and Y-GATA-H4) and five additional STRs (DYS388, DYS446, DYS447, DYS449 and DYS464), and Y chromosomal haplogroup distribution in 270 unrelated individuals from the Pathans residing in the Federally Administered Tribal Areas and the North-West Frontier Province of Pakistan using in-house multiplex PCR systems. Each Y-STR showed diversities ranging from 0.2506 to 0.8538, and the discriminatory capacity (DC) was 73.7% with 199 observed haplotypes using 17 Yfiler loci. By the addition of 5 Y-STRs to the Yfiler system, the DC was increased to 85.2% while showing 230 observed haplotypes. Among the additional 5 Y-STRs, DYS446, DYS447 and DYS449 were major contributors to enhancing discrimination. In the analysis of molecular variance, the Pathans of this study showed significant differences from other Pathan populations as well as neighboring population sets. In Y-SNP analysis, a total of 12 Y chromosomal haplogroups were observed and the most frequent haplogroup was R1a1a with 49.3% frequency. To obtain insights on the origin of Pathans, the network analysis was performed for the haplogroups G and Q observed from the Pathans and the Jewish population groups including Ashkenazim and Sephardim, but little support for a Jewish origin could be found. In the present study, we report Y-STR population data in Pathans of Pakistan, and we emphasize the need for adding additional markers to the commonly used 17 Yfiler loci to achieve more improved discriminatory capacity in a population with low genetic diversity.

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Y-SNP miniplexes for East Asian Y-chromosomal haplogroup determination in degraded DNA

Abstract

Introduction

Section snippets

DNA samples

Y-SNP selection and primer design

Conclusion

Acknowledgements

Trends Genet.

Trends Genet.

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Forensic Sci. Int.

Am. J. Hum. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int.

Forensic Sci. Int. Genet.

Biochim. Biophys. Acta

Invest. Genet.

The Y chromosome in forensic analysis and paternity testing

Int. J. Legal Med.

Y chromosome sequence variation and the history of human populations

Nat. Genet.

Hierarchical patterns of global human Y-chromosome diversity

Mol. Biol. Evol.

The human Y chromosome: an evolutionary marker comes of age

Nat. Rev. Genet.

The phylogeography of Y chromosome binary haplotypes and the origins of modern human populations

Ann. Hum. Genet.