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The population genetics characteristics of Ion AmpliSeq™ MH-74 plex microhaplotype research panel

  • Shengqiu Qu
    Correspondence
    Corresponding author at: Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, China.
    Affiliations
    Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, China

    Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Cologne, Germany
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  • Author Footnotes
    † We would like to dedicate this paper to Peter M. Schneider, who unfortunately passed away just before the paper was submitted for publication. He played an essential role in the research described here and he will be sincerely missed.
    Peter M. Schneider
    Footnotes
    † We would like to dedicate this paper to Peter M. Schneider, who unfortunately passed away just before the paper was submitted for publication. He played an essential role in the research described here and he will be sincerely missed.
    Affiliations
    Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Cologne, Germany
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  • Robert Lagacé
    Affiliations
    Human Identification Division HID – MPS, Thermo Fisher Scientific, South San Francisco, USA
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  • Maximilian Neis
    Affiliations
    Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Cologne, Germany
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  • Weibo Liang
    Affiliations
    Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, China
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  • Lin Zhang
    Affiliations
    Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, China
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  • Author Footnotes
    † We would like to dedicate this paper to Peter M. Schneider, who unfortunately passed away just before the paper was submitted for publication. He played an essential role in the research described here and he will be sincerely missed.
Published:October 25, 2022DOI:https://doi.org/10.1016/j.fsigss.2022.10.068

      Abstract

      A large number of microhaplotype markers have been investigated for various forensic purposes in recent years. In the present study, we have analysed population genetic data and sequencing performance of the Ion AmpliSeq™ MH-74 plex microhaplotype research panel. A total of 178 samples from 3 major biogeographic regions including 54 Africans, 61 Iraqis from the Middle East and 63 Germans were genotyped using this panel on the Ion S5™ MPS platform (Thermo Fisher Scientific). Then we obtained the allele frequencies, the effective number of alleles (Ae), and the forensic efficiency parameters of the 74 MHs. Finally, STRUCTURE analysis was used to evaluate and compare the biostatistical efficiency of the 74 MHs in the 3 major biogeographic regions. All results demonstrated that the Ion AmpliSeq™ MH-74 plex microhaplotype research panel is characterized by a reliable sequencing performance.

      Keywords

      1. Introduction

      Microhaplotypes (MHs) are novel compound genetic markers of two or more SNPs within a short distance (less than 300 bp) [
      • Kidd K.K.
      • Pakstis A.J.
      • Speed W.C.
      • Lagace R.
      • Chang J.
      • Wootton S.
      • Haigh E.
      • Kidd J.R.
      Current sequencing technology makes microhaplotypes a powerful new type of genetic marker for forensics.
      ,
      • Kidd K.K.
      • Pakstis A.J.
      State of the art for microhaplotypes.
      ]. After the exploration of many years, the massively parallel sequencing (MPS) technology which enables the clonal sequencing of parental haplotypes on the paternal and maternal chromosomes is chosen to genotype this new marker. MHs have several advantages compared to short tandem repeats (STRs), such as low mutation rates, lack of stutter artifacts, short amplicons, and non-preferential amplification of each allele and also more polymorphic than single nucleotide polymorphisms (SNP), which have improved the capabilities of human identification, kinship analysis, mixture deconvolution, and ancestry prediction [
      • Oldoni F.
      • Kidd K.K.
      • Podini D.
      Microhaplotypes in forensic genetics.
      ]. In recent years, a large number of microhaplotype markers based on the MPS technology have been investigated for various forensic purposes have been published. For forensic applications, it is important to provide reference population allele frequencies detected by MPS direct sequencing and make them available to the global forensic community. The Ion AmpliSeq MH-74 Plex Research Panel (MH-74 panel) is a 157 – 325 bp assay covering 74 microhaplotypes (230 SNPs) selected from a set of 130 microhaplotypes previously characterized by the Kidd Laboratory [
      • Kidd K.K.
      • Speed W.C.
      • Pakstis A.J.
      • Podini D.S.
      • Lagace R.
      • Chang J.
      • Wootton S.
      • Haigh E.
      • Soundararajan U.
      Evaluating 130 microhaplotypes across a global set of 83 populations.
      ]. It was described in a 2020 publication by Oldoni, et al. [
      • Oldoni F.
      • Bader D.
      • Fantinato C.
      • Wootton S.C.
      • Lagace R.
      • Kidd K.K.
      • Podini D.
      A sequence-based 74plex microhaplotype assay for analysis of forensic DNA mixtures.
      ]. In the present study, we have analysed the population genetic data and sequencing performance of the MH-74 panel.

      2. Material and methods

      A total of 178 samples from 3 major biogeographic regions including 54 Africans (26 from West Africa and 28 from East Africa), 61 Iraqis from the Middle East and 63 Germans were collected from individuals who self-identified as belonging to one of those groups with written informed consent. DNA samples were amplified for the Ion AmpliSeq™ MH-74 plex microhaplotype research panel on the GeneAmp® 9700 System (Thermo Fisher Scientific) according to the manufacturer’s protocol and then sequenced on the Ion S5™ MPS platform (Thermo Fisher Scientific). Sequencing data were processed by the Torrent Suite™ Software and the HID-Microhaplotype-Research-PluginV1.5 (Thermo Fisher Scientific). STRAF 1.0.5 (STR Analysis for Forensics) online tool was used to calculate forensic parameters including Gene Diversity (GD), power of discrimination (PD), probability of exclusion (PE), and typical paternity index (TPI). Then we obtained the effective number of alleles (Ae) based on the formula proposed by Kidd and Speed. STRUCTURE v. 2.3.4 was used to evaluate and illustrate the population genetic structure of our dataset. Each run consisted of 5 iterations of 10,000 burn-in steps followed by 10,000 MCMC steps to achieve accurate estimation of posterior probabilities with K values ranging from 2 to 6. The STRUCTURE HARVESTER was used to confirm the optimum K value with results of STRUCTURE.

      3. Results and discussion

      The allele frequencies of these 74 MHs demonstrated high polymorphism in all the unrelated samples. There are three microhaplotypes (mh06KK-008, mh10KK-169, and mh13KK-218) showing 15 haplotypes in all 178 individuals. The effective number of alleles (Ae) was calculated based on the formula proposed by Kidd and Speed. Ae values ranged between 1.02 and 8.48 for 74 MHs, and the mean values of Ae for these 74 loci were 3.36. As with the number of haplotypes, mh13KK-218 has the highest Ae value. The distribution values of relevant forensic statistical parameters of the 74 MHs are shown in Fig. 1. The GD values ranged between 0.02 and 0.88, and the mean values of GD for these 74 loci were 0.66. The PD values ranged between 0.04 and 0.97, and the mean values of PD for these 74 loci were 0.81.
      Fig. 1
      Fig. 1Forensic statistical parameters and Ae value calculated for the 74 MHs in all the unrelated samples.
      The results of STRUCTURE analysis of the 3 major biogeographic populations are presented in Fig. 2. Each individual is represented by a vertical line partitioned into K colored segments. According to the rate of change of the likelihood distribution (mean), the optimal K value was 3. At K= 3, three major distinct composition were obtained from the panel. In addition, East Africa and West Africa also showed different ancestry composition. The four ancestral clusters were East Africa, West Africa, Middle East and Europe.
      Fig. 2
      Fig. 2STRUCTURE analysis of 74 MHs for 3 major biogeographic populations from K= 2 to K= 6.

      4. Conclusion

      We report on allele frequency data, genetic relationship and differences using the 74-MH panel in three major biogeographic regions from Sub-Saharan Africa, Middle East and Europe. Especially population genetics data of the Middle East demonstrate a clear separation from African and European samples which is not easily possible based on single SNP data. All results demonstrate that the Ion AmpliSeq™ MH-74 plexmicro-haplotype research panel is characterized by a reliable sequencing performance. It exhibits a strong power of exclusion in forensic scenarios and offers a potential application for ancestry inference.

      Funding

      These studies have been supported in part by China Scholarship Council.

      Ethics statement

      The studies involving human participants were reviewed and approved by the Ethics Commission of Cologne University’s Faculty of Medicine. The participants provided their written informed consent to participate in this study.

      Acknowledgments

      Thanks to all the researchers at the Institute of Legal Medicine at the Faculty of Medicine, University of Cologne. We would also like to thank Dr. Fabio Oldoni for his assistance with our study.

      Conflict of interest statement

      None.

      References

        • Kidd K.K.
        • Pakstis A.J.
        • Speed W.C.
        • Lagace R.
        • Chang J.
        • Wootton S.
        • Haigh E.
        • Kidd J.R.
        Current sequencing technology makes microhaplotypes a powerful new type of genetic marker for forensics.
        Forensic Sci. Int. Genet. 2014; 12: 215-224
        • Kidd K.K.
        • Pakstis A.J.
        State of the art for microhaplotypes.
        Genes. 2022; 13
        • Oldoni F.
        • Kidd K.K.
        • Podini D.
        Microhaplotypes in forensic genetics.
        Forensic Sci. Int. Genet. 2019; 38: 54-69
        • Kidd K.K.
        • Speed W.C.
        • Pakstis A.J.
        • Podini D.S.
        • Lagace R.
        • Chang J.
        • Wootton S.
        • Haigh E.
        • Soundararajan U.
        Evaluating 130 microhaplotypes across a global set of 83 populations.
        Forensic Sci. Int. Genet. 2017; 29: 29-37
        • Oldoni F.
        • Bader D.
        • Fantinato C.
        • Wootton S.C.
        • Lagace R.
        • Kidd K.K.
        • Podini D.
        A sequence-based 74plex microhaplotype assay for analysis of forensic DNA mixtures.
        Forensic Sci. Int. Genet. 2020; 49102367