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Analysis of 124 SNP loci included in HID Ampliseq identity panel in a small population of Rio de Janeiro, Brazil

  • C.G. Bottino
    Affiliations
    Instituto de Pesquisa e Perícias em Genética Forense (IPPGF), DGPTC/PCERJ, Rio de Janeiro, Brazil

    Instituto Nacional de Metrologia, Qualidade e Tecnologia (Inmetro), Duque de Caxias, Brazil
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  • R. Silva
    Affiliations
    Instituto de Biofísica Carlos Chagas Filho/UFRJ, Rio de Janeiro, Brazil
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  • R.S. Moura-Neto
    Correspondence
    Corresponding author at: Instituto de Biologia/UFRJ, Av. Carlos Chagas Filho, 373, Bloco A, Sala A0-087, Cidade Universitária, Rio de Janeiro, CEP 21.941-902, RJ, Brazil.
    Affiliations
    Instituto Nacional de Metrologia, Qualidade e Tecnologia (Inmetro), Duque de Caxias, Brazil

    Instituto de Biologia/UFRJ, Rio de Janeiro, Brazil
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Published:September 27, 2019DOI:https://doi.org/10.1016/j.fsigss.2019.09.093

      Abstract

      Implementation of massively parallel sequencing platforms can bring a great contribution to Forensic Genetics field, with a great saving of time and costs, as well as allowing reliable results to be obtained from small or extremely degraded samples. The aim of this work was to analyze 124 SNP loci (90 autosomal and 34 Y-SNP) included in HID-Ion Ampliseq Identity Panel in a small sample from Rio de Janeiro state, Brazil. Samples from 12 non-related individuals were amplified with HID-Ion Ampliseq Identity Panel and sequenced on the Ion Torrent PGM platform (Thermo Fisher Scientific); genotypes were generated with HID SNP Genotyper plugin and forensic parameters were calculated with PowerStats v.12. All samples were successfully genotyped and were used to calculate allele frequencies, homozygosity, heterozygosity, random match probability (RMP) and exclusion power for all 90 autosomal SNP loci. Using the formula proposed by Budowle et al. (1996), only 4 of the 90 loci genotyped (4,4%) showed allele frequencies below the minimum required. It means that although a small set of individuals was used on this study, it may have shown a good perspective of Rio de Janeiro state allele frequencies. Among the 11 male samples analyzed, a prevalence of the haplogroup R1b of Y chromosome was observed, followed by the haplogroups E, Q and J. Such distribution reflects the results demonstrated in other studies for the population of Rio de Janeiro. All results together demonstrate the usefulness and applicability of SNP analysis on Ion Torrent PGM.

      Keywords

      1. Introduction

      Massively parallel sequencing (MPS) platforms allow the simultaneous analysis of thousands to millions of DNA fragments, generating large amounts of data in a relatively short time frame compared with traditional sequencing methods. Implementation of such analysis would allow reliable results to be obtained from small or extremely degraded samples [
      • De Knijff P.
      From next generation sequencing to now generation sequencing in forensics.
      ]. First applications of MPS on Forensic Genetics were published in early 2010’s with single nucleotide polymorphisms (SNPs) analysis [
      • Børsting C.
      • et al.
      Evaluation of the Ion TorrentTM HID SNP 169-plex: a SNP typing assay developed for human identification by second generation sequencing.
      ,
      • Keating B.
      • et al.
      First all-in-one diagnostic tool for DNA intelligence: genome-wide inference of biogeographic ancestry, appearance, relatedness, and sex with the Identitas v1 Forensic Chip.
      ,
      • Seo S.B.
      • et al.
      Single nucleotide polymorphism typing with massively parallel sequencing for human identification.
      ].
      SNPs are point mutations on DNA (substituions, insertions or deletions) that are usefull on genotyping degraded samples, as they require smaller fragments to be amplified and analysed. They are distributed throughout the human genome and can be divided in 4 categories: identity, lineage informative, ancestry informative or fenotipic [
      • Budowle B.
      • Van Daal A.
      Forensically relevant SNP classes.
      ]. A panel of 124 SNP loci (90 autossomal and 34 Y-SNPs) were mapped by Kidd et al [
      • Kidd K.K.
      • et al.
      Developing a SNP panel for forensic identification of individuals.
      ] and the SNPforID consortium [
      • Sanchez J.J.
      • et al.
      A multiplex assay with 52 single nucleotide polymorphisms for human identification.
      ] and incorporated on HID-Ion AmpliSeq Identity Panel™ (Thermo Fisher Scientific), a multiplex kit for genotyping samples on Ion Torrent PGM.
      The aim of this study was to analyze 124 SNP loci (90 autosomal and 34 Y-SNP) included in HID-Ion Ampliseq Identity Panel in a small sample from the state of Rio de Janeiro, Brazil.

      2. Methodology

      Buccal swabs from 12 non-related individuals from Rio de Janeiro state were collected on IPPGF/PCERJ. DNA was extracted by organic [
      • Butler J.M.
      Forensic DNA Typing.
      ] or Chelex® method [
      • Walsh P.S.
      • et al.
      Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material.
      ] and quantified by qPCR with Quantifiler HP kit (Thermo Fisher Scientific). DNA amplification was performed with HID-Ion Ampliseq Identity Panel™ (Thermo Fisher Scientific), followed by library preparation with HID-Ion Ampliseq Library ™ kit and Ion Xpress Barcode Adapters™ kit (Thermo Fischer Scientific). Emulsion PCR and enrichment was performed with Ion PGM™ Hi-Q™ OT2 kit (Thermo Fischer Scientific) and the libraries were sequenced in Ion Torrent PGM™ with Ion PGM™ Hi-Q™ Sequencing kit and chip 318 v2 (Thermo Fischer Scientific).
      Genotypes were generated with HID SNP Genotyper plugin and forensic parameters were calculated with PowerStats v.12. Estimation of minimum allele frequencies was performed as described by Budowle et al [
      • Budowle B.
      • et al.
      Estimating minimum allele frequencies for DNA profile frequency estimates for PCR-based loci.
      ].

      3. Results and discussion

      All samples were successfully genotyped and were used to calculate allele frequencies and forensic parameters for all 90 autosomal SNP loci (Table 1). Using the formula proposed by Budowle et al [
      • Budowle B.
      • et al.
      Estimating minimum allele frequencies for DNA profile frequency estimates for PCR-based loci.
      ], only 4 of the 90 loci genotyped (4,4%) showed allele frequencies below the minimum required. It was possible to verify that although a small set of individuals was used on this study, it may have shown a good perspective of Rio de Janeiro state allele frequencies. Results are also similar to those published in a previous study for Brazilian population [
      • Avila E.
      • et al.
      Forensic characterization of Brazilian regional populations through massive parallel sequencing of 124 SNPs included in HID ion Ampliseq Identity Panel.
      ].
      Table 1Forensic parameters for a sample of 12 individuals from Rio de Janeiro state based on 90 autosomal SNPs included in HID-Ion Ampliseq Identity Panel.
      ParameterValues
      Allele frequencies (range)0.042–0.958
      Homozygosity (mean; min – max)0.453 (0.083–0.833)
      Heterozygosity (mean; min – max)0.450 (0.083–0.522)
      Random Match Probability (RMP) (combined; min – max)7.643 × 10−32 (0.333–0.847)
      Power of Exclusion (PE) (combined; min – max)0.999999998518661 (0.006–0.662)
      Among the 11 male samples analyzed, a prevalence of the haplogroup R1b of Y chromosome was observed, followed by the haplogroups E, Q and J (Table 2). Such distribution reflects the results demonstrated in other studies for the population of Rio de Janeiro state [
      • Resque R.
      • et al.
      Male lineages in Brazil: intercontinental admixture and stratification of the European background.
      ].
      Table 2Frequencies of Y haplogroups found in a set of 11 individuals from Rio de Janeiro state genotyped with HID-Ion Ampliseq Identity Panel.
      Haplogroup#Biogeographical origin
      R1b5 (45.5%)European
      E3 (27.3%)African / Mediterranean
      Q2 (18.2%)Native American
      J1 (9.0%)European / Middle East

      4. Conclusion

      All results together demonstrate the usefulness and applicability of SNP analysis on Ion Torrent PGM for Rio de Janeiro state population.

      Role of funding

      CNPq, Capes, and FAPERJ.

      Declaration of Competing Interest

      None.

      Acknowledgements

      The authors would like to thank Thermo Fischer Scientific for providing the HID Ampliseq Identity Panel and technical support; and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Grant No 312905/2018-1), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES Grant No. 23038.006844/2014-46), and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ Grant No. 2002.532/2019) for the financial support.

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