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