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Multiplex Single-Nucleotide Polymorphism Typing of the Human Y Chromosome Using Taqman Probes

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Multiplex Single-Nucleotide Polymorphism Typing of the Human Y Chromosome Using Taqman Probes University of Pennsylvania ScholarlyCommons Department of Anthropology Papers Department of Anthropology 5-31-2011 Multiplex Single-Nucleotide Polymorphism Typing of the Human Y Chromosome Using TaqMan Probes Begoña Martínez-Cruz Janet Ziegle Paula Sanz Graciela Sotelo Roger Anglada See next page for additional authors Follow this and additional works at: https://repository.upenn.edu/anthro_papers Part of the Anthropology Commons, and the Genetics and Genomics Commons Recommended Citation Martínez-Cruz, B., Ziegle, J., Sanz, P., Sotelo, G., Anglada, R., Plaza, S., Comas, D., Genographic Consortium, & Schurr, T. G. (2011). Multiplex Single-Nucleotide Polymorphism Typing of the Human Y Chromosome Using TaqMan Probes. Investigative Genetics, 2 13. https://doi.org/10.1186/2041-2223-2-13 Theodore G. Schurr is not listed as an individual author on this paper but is part of the Genographic Consortium. A full list of Genographic Consortium members for this paper can be found in the Acknowledgements. This paper is posted at ScholarlyCommons. https://repository.upenn.edu/anthro_papers/49 For more information, please contact [email protected]. Multiplex Single-Nucleotide Polymorphism Typing of the Human Y Chromosome Using TaqMan Probes Abstract Background The analysis of human Y-chromosome variation in the context of population genetics and forensics requires the genotyping of dozens to hundreds of selected single-nucleotide polymorphisms (SNPs). In the present study, we developed a 121-plex (121 SNPs in a single array) TaqMan array capable of distinguishing most haplogroups and subhaplogroups on the Y-chromosome human phylogeny in Europe. Results We present data from 264 samples from several European areas and ethnic groups. The array developed in this study shows >99% accuracy of assignation to the Y human phylogeny (with an average call rate of genotypes >96%). Conclusions We have created and evaluated a robust and accurate Y-chromosome multiplex which minimises the possible errors due to mixup when typing the same sample in several independent reactions. Keywords Y-chromosome variation, single-nucleotide polymorphisms, SNPs Disciplines Anthropology | Genetics and Genomics | Social and Behavioral Sciences Comments Theodore G. Schurr is not listed as an individual author on this paper but is part of the Genographic Consortium. A full list of Genographic Consortium members for this paper can be found in the Acknowledgements. Author(s) Begoña Martínez-Cruz, Janet Ziegle, Paula Sanz, Graciela Sotelo, Roger Anglada, Stéphanie Plaza, David Comas, Genographic Consortium, and Theodore G. Schurr This journal article is available at ScholarlyCommons: https://repository.upenn.edu/anthro_papers/49 Martínez-Cruz et al. Investigative Genetics 2011, 2:13 http://www.investigativegenetics.com/content/2/1/13 RESEARCH Open Access Multiplex single-nucleotide polymorphism typing of the human Y chromosome using TaqMan probes Begoña Martínez-Cruz1, Janet Ziegle2, Paula Sanz1, Graciela Sotelo1, Roger Anglada3, Stéphanie Plaza3 and David Comas1* for the Genographic Consortium Abstract Background: The analysis of human Y-chromosome variation in the context of population genetics and forensics requires the genotyping of dozens to hundreds of selected single-nucleotide polymorphisms (SNPs). In the present study, we developed a 121-plex (121 SNPs in a single array) TaqMan array capable of distinguishing most haplogroups and subhaplogroups on the Y-chromosome human phylogeny in Europe. Results: We present data from 264 samples from several European areas and ethnic groups. The array developed in this study shows >99% accuracy of assignation to the Y human phylogeny (with an average call rate of genotypes >96%). Conclusions: We have created and evaluated a robust and accurate Y-chromosome multiplex which minimises the possible errors due to mixup when typing the same sample in several independent reactions. Background The human Y chromosome has been extensively ana- The development of high-throughput technologies to lysed in forensic and evolutionary studies because of its genotype hundreds of thousands of markers has yielded unique properties. Despite being a complex chromosome an increase in the understanding of the genetic diversity with highly repetitive sequences [6], its exclusively paternal of our species [1]. This genomic knowledge has been inheritance due to the lack of recombination over most of applied to different fields, from biomedical and pharma- the chromosome has allowed researchers to trace paternal ceutical research to population genetics and forensics lineages and reconstruct the male demographic history of [2-5]. Most of the genotyping technologies have been populations [7]. The human Y chromosome contains hun- based on typing of single-nucleotide polymorphisms dreds of well-characterised SNPs (around 600) whose evo- (SNPs) that commonly have only two alleles (ancestral or lutionary relationships have been established in a robust derived compared to nonhuman primates) and have phylogeny [8]. The Y-chromosome phylogeny defines sev- usually arisen once. These high-throughput SNP geno- eral main branches (haplogroups) named with alphabetical typing analyses provide much information about the var- (A to T) and numerical codes. Each branch is subdivided iation of our genome, but still little information has been into more minor braches (subhaplogroups) defined by derived from some specific genome regions of great other SNPs in a hierarchical way [9]. The classification of interest. SNPs are also used for human identification pur- a male DNA sample within the Y-chromosome phylogeny poses and to reconstruct human demographic history, requires the successive typing of markers that define the although these fields require the typing of a few selected haplogroup and subhaplogroups to which the sample SNPs for targeted research rather than the use of high- belongs. This process is not only laborious but also time- throughput genotyping methods (that is, allele-specific consuming. For this reason, several attempts to genotype probes or single base primer extensions). multiple Y-chromosome SNPs in a single reaction have been reported, mainly using single-base extension meth- odologies [10-15] or oligonucleotide ligation assays [16]. * Correspondence: [email protected] 1Institut de Biologia Evolutiva (CSIC-UPF), Departament de Ciències Nonetheless, these multiplex approaches have not been Experimentals i de la Salut, Universitat Pompeu Fabra, Doctor Aiguader 88, able to type successfully more than 35 Y-chromosome E-08003 Barcelona, Spain SNPs in a single reaction. Full list of author information is available at the end of the article © 2011 Martínez-Cruz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Martínez-Cruz et al. Investigative Genetics 2011, 2:13 Page 2 of 7 http://www.investigativegenetics.com/content/2/1/13 It is preferable to genotype, in a reproducible manner, present in the populations typed and 24 of which were around 100 SNPs at a time. We therefore aimed to design shared by two or more European samples (Figure 2). a single multiplex reaction of this size using TaqMan Haplogroup composition varied greatly across the differ- probes (Applied Biosystems, Inc., Foster City, CA, USA). ent populations sampled, as expected from the known TaqMan probes are robust and reproducible assays that phylogeography of the samples used. have been used extensively to type SNPs in single reac- Of 282 individuals where genotyping was attempted, tions, that is, one SNP per TaqMan reaction. Each assay three did not yield any amplified product for any assay. is based on TaqMan probes that are hydrolysis probes This failure was due to technical manipulation problems that anneal within a specific DNA region amplified by in the DNA loading phase, as the wells corresponding polymerase chain reactions (PCRs). The probes present a to these three samples were empty in the array. Of the fluorophore (usually VIC or 6-FAM, Applied Biosystems) remaining 279, 277 showed complete phylogenetic com- attached to the 5′-end and a quencher at the 3′-end that patibility. If a sample showed the derived allele for a prevents fluorescence of the fluorophore. As the Ampli- SNP that led to a certain branch of the tree, it also pre- Taq Gold DNA Polymerase (Applied Biosystems) extends sented the derived alleles for all the SNPs leading to the primer, its exonuclease activity degrades the probe that branch and the ancestral alleles for the SNPs lead- that has annealed to the template and releases the fluoro- ing to other branches. For example, if a sample was phore, relieving the quenching effect and allowing fluor- derived for M201 (allele T), it was also derived for M89 escence of the fluorophore. In a TaqMan assay, two (allele T), M168 (allele T), M42 (allele T) and SRY10831.1 probes labelled with different fluorophores are used, each (allele G), but was ancestral for all the rest of the SNPs one complementary to one of the two alleles (ancestral in the array (see Table 1). Although some of the hap- or derived) of a SNP. In this way, in a real-time PCR, the logroups (that is, haplogroups D and M) were not pre- alleles of one SNP of a DNA sample can be interrogated sent in the populations sampled, all the samples
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