Estimation of the number of contributors to mixed samples of DNA by mitochondrial DNA analyses using massively parallel sequencing

  • Hiroaki NakanishiEmail author
  • Koji Fujii
  • Hiroaki Nakahara
  • Natsuko Mizuno
  • Kazumasa Sekiguchi
  • Katsumi Yoneyama
  • Masaaki Hara
  • Aya Takada
  • Kazuyuki Saito
Original Article


We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209–16,400) and B- (positions 30–284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.


Number of contributors Mixed DNA Mitochondrial DNA Massively parallel sequencing 



The authors thank Dr. Hajime Miyaguchi (National Research Institute of Police Science) for his help with the MiSeq analyses.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Informed consent

Informed consent was obtained from all of the participants.

Ethics approval

This study was approved by the Ethics Committee of Juntendo University School of Medicine (No. 2016134).

Supplementary material

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Hiroaki Nakanishi
    • 1
    Email author
  • Koji Fujii
    • 2
  • Hiroaki Nakahara
    • 2
  • Natsuko Mizuno
    • 2
  • Kazumasa Sekiguchi
    • 2
  • Katsumi Yoneyama
    • 3
  • Masaaki Hara
    • 3
  • Aya Takada
    • 3
  • Kazuyuki Saito
    • 1
  1. 1.Department of Forensic MedicineJuntendo University School of MedicineTokyoJapan
  2. 2.National Research Institute of Police ScienceChibaJapan
  3. 3.Department of Forensic MedicineSaitama Medical UniversitySaitamaJapan

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