International Journal of Legal Medicine

, Volume 132, Issue 2, pp 343–352 | Cite as

An SNP panel for the analysis of paternally inherited alleles in maternal plasma using ion Torrent PGM

  • Donggui Yang
  • Hao Liang
  • Shaobin Lin
  • Qing Li
  • Xiaoyan Ma
  • Jun Gao
  • Hongyu Sun
  • Qingqing Chen
  • Jianzhu Wu
  • Xueling Ou
Original Article


Researchers have sought to develop an effective protocol for paternity analysis using cell-free DNA (cfDNA) in maternal plasma. The use of massively parallel sequencing (MPS) technology for SNP testing is attractive because of its high-throughput capacity and resolution to single-base precision. In this study, we designed a customized SNP panel for cfDNA sequencing that includes 720 short amplicons (< 140 bp) targeting SNPs on the autosome and Y chromosome. The systemic performance was evaluated using the Ion Torrent PGM, indicating balanced coverage among most of the included loci, except for 78 poorly performing SNPs that were observed to have an inconsistent allele balance, lower coverage reads or high background signals. Then, the custom panel was used to perform cfDNA genotyping in maternal plasma from 20 pregnancies in the first and second trimesters (9 to 21 weeks). By establishing an allele fraction cutoff of 2.0%, 53 to 128 autosomal SNP loci were considered informative for paternal origin. Validation results in foetal samples showed that 49.43% to 100% of the real paternal alleles were accurately identified, with incorrect alleles encountered in 3 cases. The concentration of foetal cfDNA ranged from 4.28% to 10.70%. Our results show that this amplicon-based sequencing strategy could be utilized in analysing paternally inherited alleles in maternal plasma. However, further studies and optimization are required for a more detailed and accurate interpretation of the cfDNA sequencing results based on MPS technology.


Massively parallel sequencing (MPS) Single-nucleotide polymorphism (SNP) Cell-free DNA (cfDNA) Maternal plasma Paternally inherited allele 



This study was supported by grants from the National Natural Science Foundation of People’s Republic of China (Grants 81471825 and 81001352) and the Fundamental Research Funds for the Central Universities (12YKPY04). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors wish to thank Qing Yang and Yong Chen at Thermo Fisher Scientific for the technical supports.

Compliance with ethical standards

The authors declare that they have no conflict of interest. All procedures performed in studies involving human participants were in accordance with the ethical standards of the Human Subjects Committee at the Zhongshan School of Medicine, Sun Yat-sen University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Supplementary material

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Donggui Yang
    • 1
  • Hao Liang
    • 1
  • Shaobin Lin
    • 2
  • Qing Li
    • 3
  • Xiaoyan Ma
    • 3
  • Jun Gao
    • 4
  • Hongyu Sun
    • 1
  • Qingqing Chen
    • 3
  • Jianzhu Wu
    • 2
  • Xueling Ou
    • 1
  1. 1.Faculty of Forensic Medicine, Zhongshan School of MedicineSun Yat-sen UniversityGuangzhouPeople’s Republic of China
  2. 2.Fetal Medicine Center, Department of Obstetrics and GynecologyThe First Affiliated Hospital of Sun Yat-sen UniversityGuangzhouPeople’s Republic of China
  3. 3.Forensic Identification Institute of The Third Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education InstitutesGuangzhouPeople’s Republic of China
  4. 4.Reproductive Medicine CenterThe First Affiliated Hospital of Sun Yat-sen UniversityGuangzhouPeople’s Republic of China

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